Recombinant DNA processes using a dNTP mixture containing modified nucleotides

ABSTRACT

Amplification mixtures, kits, amplicons, kits and processes are provided for amplifying a nucleic acid. In particular, provided are processes which utilize an amplification mixture comprising a polymerase, a deoxynucleotidetriphosphate (DNTP) mixture which contains modified dNTPs, a first primer and a second primer. Further provided are DNTP mixtures which contain modified dNTPs for at least two of the four nucleotide triphosphates, which when incorporated into a polynucelotide, impart resistance to enzymatic degradation by an exonuclease at the sites of incorporation of the modified dNTPs. Also provided are the amplicons and vectors which incorporate the modified nucleotides.

This application is a division of U.S. patent application Ser. No.10/002,292, filed Nov. 15, 2001, which claims priority to U.S.provisional patent application Ser. No. 60/325,612 filed Sep. 28, 2001,the entire contents of which are incorporated herein by reference.

FIELD OF INVENTION

The present invention relates to recombinant DNA processes using adeoxynucleotide triphosphate (DNTP) mixture containing modified dNTPs,which when incorporated into a polynucleotide, impart resistance againstenzymatic degradation by an exonuclease at the sites of incorporation ofthe modified dNTPs.

BACKGROUND OF THE INVENTION

Many widely known recombinant DNA techniques involve replicating oramplifying DNA. One such example is the cloning of an insert DNA into atarget DNA fragment. During this procedure, the target fragment istypically digested with a restriction enzyme such as EcoRI. Similarly,the insert DNA, having the gene of interest, is digested with the sameenzyme. In one type of restriction enzyme digestion, cleavage of boththe target DNA and insert DNA leaves overlapping 3′ or 5′ nucleotidefragments on each end. These cohesive, overlapping fragments or “stickyends” are well-known properties of some restriction enzymes. Incubationof the target and insert DNA together at an appropriate temperatureallows the insert DNA to noncovalently bind to the target DNA. Thetarget DNA and insert DNA are held together by hydrogen bonding of thecohesive ends. Further incubation with an enzyme, such as DNA ligase,results in ligation of the insert DNA to the target nucleotide strand.

Another method of adding an insert nucleotide fragment into a target DNAis known as blunt-end ligation. Digestion with some restriction enzymes,such as SrfI (GCCC/GGGC), SmaI (CCC/GGG), or Eco RV (GAT/ATC) do notleave any 3′ or 5′ overhanging nucleotides at the enzyme splice site.These enzymes are known as “blunt-end” enzymes due to this feature oftheir enzymatic activity. After digestion, blunt-end restriction enzymesmaintain single 5′ “terminal” phosphates on both sides of therestriction site. These terminal 5′ phosphates are required by DNAligase for any subsequent religation of the digested DNA sequence.

The ExoClone™-PCR Cloning Kit (Sigma, St. Louis, Mo.) utilizes a methodby which multibase sticky end ligations between PCR products andsuitably cleaved plasmid DNA is accomplished. Amplified DNA inserts areproduced using specially designed primers whose 5′ ends are cohesivewith EcoRI cleaved vectors, i.e., 5′ pAATTC. A modified mix containingthiodeoxyguanosine triphosphate (sdGTP) and deoxyguanosine triphosphate(dGTP) is used to amplify high GC and long (up to 4 kb) targets. Becauseexonuclease III cleaves phosphorothioates extremely slowly, if at all,digestion with exonuclease III exposes the bases of the 5′ termini whichare cohesive with the EcoRI digested vectors. This method is limited tocloning DNA into EcoRI cleaved vectors because the EcoRI recognitionsite is the only commonly used restriction site whose 5′ four baseoverhang is punctuated by a base, i.e., guanine, not represented withinthe 5′ PAATTC overhang.

Several methods have been devised for preferentially cloning insert DNAfragments into target sequences in one orientation. These methods arecommonly known as directional cloning techniques and have been devisedto position genes in the correct 5′→3′ orientation. Directional cloningis commonly performed by digesting the target nucleotide sequences withtwo different restriction enzymes. This method results in a moleculewith dissimilar DNA ends at the target insertion site. The insert DNA isthen digested with the same two restriction enzymes thereby having twodissimilar DNA ends that correspond to a specific orientation in thetarget insertion site. By following this procedure, the insert DNA onlybinds to the target sequence in one orientation.

Another method of directionally cloning an insert into a target sequenceuses an exonuclease, such as Exonuclease III, to create the “stickyends”. See Kaluz, et al., Nucleic Acids Research, 1992, 20:4369-4370;U.S. Pat. Nos. 5,580,759, 5,518,901, 5,688,669 and 5,744,306. In thismethod, insert DNA fragments are digested with exonuclease III, a doublestrand specific exonuclease that catalyzes the stepwise release ofnucleotides from the 3′ hydroxyl termini of double stranded DNA, toproduce cohesive ends. Digestion with exonuclease III is performed atlow temperatures for very short times, usually 30-90 seconds, in orderto prevent excessive degradation. After a timed digestion, the insertfragments have 5′ overlapping nucleotide tails. These 5′ nucleotidetails are engineered so that the 5′ ends hybridize in one orientationupon base pairing to the target plasmid DNA molecule thereby resultingin a relatively simple method of directional cloning. While the use ofexonuclease III provides a relatively simple method for directionalcloning, it may be difficult to control the length of the generatedcohesive ends which may be critical when cloning small size insert DNA.

There are several drawbacks, however, with single restriction enzymedigestion cloning methods and double restriction enzyme digestiondirectional cloning methods. For instance, digesting both the target DNAand insert DNA with restriction enzymes can be time consuming andmultiple enzyme digestions increase the risk that either the target orinsert DNA sequence will be cleaved at an internal restriction site. Itis preferable that the sequence of the target DNA be known so thatrestriction enzymes are not selected which would cleave the sequence atan internal restriction site. Additionally, some restriction enzymescleave very poorly, or not at all, when their recognition sequence is ator near the termini of a DNA strand.

Accordingly, it is desirable to formulate reagents and components foruse in cloning and other recombinant DNA methodologies which could beutilized independent of the sequence being replicated or amplified. Suchformulations would obviate the need for tedious control of restrictiondigestion and at the same time have minimal impact on PCR amplificationperformance. The provision of such process and reagent mixtures wouldavoid tedious or expensive aspects of current directional cloning suchas multiple restriction enzyme digestion, addition of extra nucleotidesto the insert and/or the use of multiple primers or linkers.

SUMMARY OF THE INVENTION

One aspect of the present invention, therefore, is the provision ofmaterials, processes and kits for synthesizing DNA from a target nucleicacid. Generally, the present invention relates to processes whichincorporate modified deoxynucleotide triphosphates (dNTPs) into anucleic acid. The incorporation of these modified dNTPs impartresistance against enzymatic degradation by an exonuclease at the siteof incorporation of the modified dNTPs thereby protecting the amplifiedproduct from complete degradation.

Briefly, the present invention is directed to a process fordirectionally ligating a nucleic acid to an adaptor sequence. In thisprocess, an amplification mixture comprising a polymerase, a dNTPmixture which comprises modified dNTPs for at least one of the fourdNTPs comprising dATP, dCTP, dGTP, dTTP and analogs thereof, a firstprimer and a second primer are used. The first primer is complimentaryto a portion of the first strand of the target nucleic acid and has afirst terminus which is complimentary to a first ligation site sequenceof the adaptor sequence. The second primer is complimentary to a portionof the second strand of the target nucleic acid and has a secondterminus which is complimentary to a second ligation site sequence ofthe adaptor sequence.

The amplification mixture is used to amplify a target nucleic acidthereby producing an amplified product, or an amplicon, from the targetnucleic acid sequence. The primers anneal to each strand of the nucleicacid and each primer is extended using a polymerase and the dNTPmixture. During the extension step of the PCR, the modified dNTPs areincorporated into the amplicons in lieu of one of the non-modifieddNTPs. The incorporation of the modified dNTPs into the amplicon impartresistance against enzymatic degradation by an exonuclease at the siteof incorporation of the modified dNTPs.

Each terminus of the amplicons is then digested with an exonuclease. Theamplicons are protected from enzymatic degradation by the exonuclease atthe sites of incorporation of the modified dNTPs and preferably, thetermini of the digested amplicons terminate at the sites ofincorporation of the modified dNTPs. After digestion with theexonuclease, the digested amplicons contain a single stranded overhangsequence at each terminus. The first single stranded overhang sequenceand the second single stranded overhang sequence are complimentary to afirst ligation site sequence and a second ligation site sequence in thefirst and second adaptor sequence, respectively.

The first or second single stranded overhang sequence of the digestedamplicons is directionally ligated to either the first or secondligation site sequence of the adaptor sequence. In a preferredembodiment, the first and second ligation site sequences are restrictionenzyme recognition sequences. After digestion with the exonuclease, thedigested amplicons have a single stranded overhang sequences which arecomplimentary to either a first or second ligation site sequence in thefirst or second adaptor sequence. In one aspect of the presentinvention, the adaptor sequence is a cloning vector and the digestedamplicons are directionally inserted into a cloning vector having aligation site which is flanked by the first and second ligation sitesequences. Preferably, at least 80% of the digested amplicons areinserted in only one direction into the cloning vector.

Another aspect of the present invention is the provision of a processfor producing amplicons using the an amplification mixture comprising apolymerase, a dNTP mixture containing modified dNTPs for at least two ofthe four nucleotide triphosphates, a first primer and a second primer.The amplicons produced using the amplification mixture have a first andsecond termini complimentary to either a first or second ligation sitesequence, and are resistant to exonuclease degradation at the sites ofincorporation of the modified dNTPs.

A further aspect of the present invention is the provision of ampliconswhich have at least two modified dNTPs incorporated therein. Theincorporation of the modified dNTPs protects the polynucleotide sequenceby imparting resistance to enzymatic degradation by an exonuclease atthe sites of incorporation of the modified dNTPs in the amplicon. Suchamplicons have a first and second termini complimentary to either afirst or second ligation site sequence of an adaptor sequence. Suchamplicons have various uses, such as for the synthesis of RNA moleculesand in vitro synthesis of proteins. Also provided are vectors containingan amplicon resistant to enzymatic degradation by an exonuclease.

A further aspect of the present invention is the provision of a kit fordirectionally ligating a nucleic acid to an adaptor sequence. These kitscontain a dNTP mixture containing modified dNTPs for at least two of thefour nucleotide triphosphates which during the extension step of thePCR, are incorporated into the amplification product in lieu of one ofthe non-modified dNTPs, and a set of instructions for using the dNTPmixture to directionally ligate a target nucleic acid to an adaptorsequence.

This dNTP mixture protects nucleotides from enzymatic degradation by anexonuclease thus increasing the efficiency and the ease of the cloningprocess. A further aspect of the present invention is to provide thedNTP mixture containing modified dNTPs for at least two of the fournucleotide triphosphates, which during the extension step of the PCR,are incorporated into the amplification product in lieu of one of thenon-modified dNTPs.

Other objects and features will be in part apparent and in part pointedout hereinafter.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a flow chart schematically illustrating the directionalcloning strategy in the present invention.

FIG. 2 is a photograph of a DNA agarose gel illustrating that productquality was independent of dNTP mixture, i.e., the product from a PCRreaction using unmodified dNTPs did not differ substantially from areaction in which alpha thiophosphorano dATPs (sdATP) and dGTPs (sdGTP)were substituted for a fraction of unmodified dATP and dGTPs. The dNTPmixture was prepared as further described in Example 1.

FIG. 3 contains graphs summarizing the amplification and protectionyields from a ³²PdNTP containing PCR. Yields were measured by TCAprecipitation. The top panel shows overall yields of amplification(incorporation) products for all enzymes normalized to Taq/sdNTP. Themiddle panel shows yields of sdNTP products normalized to dNTP. Lowerpanel shows that while all products were degraded from approximately 50%or less (+Exo/−Exo) for all the deoxynucleotide amplifications, additionof ExoIII to the thiodeoxypurine (sdPuTP) containing reactions resultedin protections ranging from approximately 80 to 250%.

FIG. 4 is a graph illustrating that nucleotide protections that aregreater than 100% could occur if the polymerase is active enough duringExoIII degradation to incorporate additional label potentially resultingin blunt-ended PCR products.

FIG. 5 contains graphs illustrating that re-incorporation during ExoIIIdigestion replaces an insignificant portion (approximately 5-15%) of thenucleotides removed by ExoIII. These data show it is safe to use thedNTP nucleotide mix with most thermostable DNA polymerases.

FIG. 6 contains graphs illustrating titration results for the fourthionucleotides. The top panel illustrates that PCR is tolerant ofthionucleotides in the order G>A>T=C. The lower panel (+ExoIII) revealsthat the amplicons are preferentially protected from ExoIII digestion inthe order G>A>T>C.

FIG. 7 is a graph illustrating the PCR reaction concentration ratios ofalpha thiophosphano dATP (sdATP) to alpha thiophosphano dGTP (sdGTP)utilized to optimize modified dNTP incorporation.

FIG. 8 contains graphs illustrating that PCR product yield begins todecrease at concentrations of thiodeoxypurine above 0.3 mM. The lowerpanel reports the occupation of purine sites by thiodeoxypurinemonophosphate (sdPuMP) as a function of dPuTP concentration.

FIG. 9 contains graphs illustrating titration results for the fourphosphoborane nucleotides (bdNTP, N=A,C,G,T). These data show that thePCR yields are negatively impacted by phosporoboranes in the orderG<C<T<A and that ExoIII protection increases in the order G>A>C=T.

FIG. 10 is a graph PCR illustrating dPu occupation as a function ofalpha boranophosphano dATP (bdATP) and alpha boranophosphano dGTP(bdGTP) concentration utilized in a PCR to optimize modified dNTPincorporation.

FIG. 11 contains graphs illustrating verification of the optimalbdATP/bdGTP ratio (bottom panel) established in FIG. 10. The useful(high protection levels) and unimpeding (little or no effect on PCRyield) concentration of bdPuTP range from approximately 0.1 to 0.3 mMbdPuTP (top panel).

FIG. 12 is a diagram illustrating the primer possibilities utilized toconfirm the directional cloning results.

FIG. 13A is a photograph of a DNA agarose gel illustrating thatamplification across the insert yielded amplicons that were larger thanfrom pUC19 and the same as an amplification product from a previouslyprepared construct containing an identical insert (lanes 1-4 vs. 5 and 6respectively).

FIG. 13B are photographs of agarose gels which demonstrate that the useof primer set EcopUC/Lambda R and HindpUC/Lambda L (FIG. 12) produced noPCR products while primer sets EcopUC/LambdaL and HindpUC/Lambda R did,thus demonstrating that all of the clones produced using EcopUC/LambdaLand HindpUC/Lambda R were directionally inserted.

FIG. 14 is a photograph of a DNA agarose gel illustrating the expected“insert” restriction fragment generated by digesting pBX withBamHI/XbaI.

FIG. 15 is a graph illustrating the expected overhang length as afunction of modified nucleotide concentration. Clearly, phosphoboranonucleotides can be used at higher cooccupation rates without impactingPCR performance.

FIG. 16 is a photograph of a DNA agarose gel illustrating the resultingligation product PCR results. All ligation reactions produced arelatively high molecular weight PCR product while only EcopUC/lambda Land HindpUC/lambda R produced products that co-migrated with theauthentic clone amplification products.

FIG. 17 is a graph illustrating the fraction duplex and the frequency ofmutation as a function of sequence position. This graph demonstratesthat there is no correlation between mutational frequency and degree ofsingle stranded overhang.

FIG. 18 is a photograph of an agarose gel which demonstrates that thethe exonuclease digested amplicons are self-ligatable. The restrictionenzyme fragment is an EcopUC/HindpUC amplified from pUC19 (i.e.,amplified across MCS), cut with BamHI (lane B), EcoRI (lane E), HindIII(lane H), or SAlI (lane S), XbaI (lane X). Lane U is the fragment whichwas not digested with ExoIII.

FIG. 19 illustrates the nucleotide sequences for the adaptor sequencesfor making linear DNA in vitro transcription/translation constructs.

FIG. 20 is a photograph of an agarose gel which demonstrates thedetection of ligation between PCR amplicon and adaptors. Lane 1 and 2are exonuclease III treated PCR products for gene p53 and IkB,respectively. Lane 3 and 4 contain amplicons from ligated p53-adaptorand IkB-adaptor constructs using primers specific for the adaptorregion.

DETAILED DESCRIPTION

All publications, patents, patent applications or other references citedin this application are herein incorporated by reference in theirentirety as if each individual publication, patent, patent applicationor reference are specifically and individually indicated to beincorporated by reference.

DEFINITIONS AND ABBREVIATIONS

To facilitate understanding of the invention, a number of terms aredefined below:

The “amplification” of nucleic acids refers to the replication of one tomany additional copies of a nucleic acid sequence by a method catalyzedby an enzyme. Preferably, it is carried out using polymerase chainreaction (PCR) technologies. A variety of amplification methods areknown in the art and are described, inter alia, in U.S. Pat. Nos.4,683,195 and 4,683,202 and in PCR Protocols: A Guide to Methods andApplications, ed. Innis et al., Academic Press, San Diego, 1990. In PCRand other primer extension methodologies, a primer refers to a shortoligonucleotide of defined sequence that is annealed to a DNA templateto provide an initiation site for a polymerase as in the polymerasechain reaction.

A “polymerase” is a catalyst, usually a protein enzyme, for forming anextension of an oligonucleotide along a DNA template where the extensionis complimentary to the template. A “polymerase” is an enzyme that iscapable of incorporating nucleoside triphosphates to extend a 3′hydroxyl group of a nucleic acid molecule, if that molecule hashybridized to a suitable template nucleic acid molecule. Polymeraseenzymes are discussed in Watson, J. D., In: Molecular Biology of theGene, 3rd Ed., W. A. Benjamin, Inc., Menlo Park, Calif. (1977), whichreference is incorporated herein by reference, and similar texts.

Exonuclease activity is, under the conditions of the reactionscontemplated herein, the catalytic control or cleavage of nucleotidesbeginning at the end of a nucleic acid. A “3′ to 5′ exonuclease”, or anenzyme having “3′ to 5′ exonuclease activity,” begins the removal orcleavage of nucleotides at the 3′ terminus of a nucleic acid andproceeds towards the 5′ end. A “5′ to 3′ exonuclease”, or an enzymehaving “5 to 3′ exonuclease activity”, begins the removal or cleavage ofnucleotides at a 5′ terminus of a nucleic acid and proceeds toward the3′ end. There are enzymes that contain either 5′ to 3′ exonucleaseactivity or 3′ to 5′ exonuclease activity, or both activities inconjunction with polymerase activities that could be used in theprocesses disclosed herein.

“Complimentary” refers to the natural association of nucleic acidsequences by base-pairing (A-G-T pairs with the complimentary sequenceT-C-A). Complementarity between two single-stranded molecules may bepartial, if only some of the nucleic acids pair are complimentary, orcomplete, if all bases pair are complimentary.

The term “recognition sequence” refers to a particular sequence which aprotein, chemical compound, DNA, or RNA molecule (e.g., restrictionendonuclease, a modification methylase, or a recombinase) recognizes andbinds. For example, a restriction enzyme recognition sequence is anucleic acid sequence which a restriction enzyme will recognize, bindand cleave.

An “adapter sequence” is any duplex nucleotide sequence suitable forcohesive ligation to one of the ends of an exonuclease digestedamplification product. Such adaptor sequences can result from enzymatic(in vivo or in vitro) or synthetic sources and can include but not belimited to cloning vehicles (plasmids, vectors, BACs etc.),amplification products and sequences prepared by non enzymatic meanssuch as solid phase DNA synthesis.

The term “cloning vector” refers to a plasmid, virus, retrovirus,bacteriophage, cosmid, artificial chromosome (bacterial or yeast), ornucleic acid sequence which is able to replicate in a host cell whichmay contain an optional marker suitable for use in the identification oftransformed cells, e.g., tetracycline resistance or ampicillinresistance. Optionally, a cloning vector may possess the featuresnecessary for it to operate as an expression vector.

The term “dNTP” refers to deoxynucleoside triphosphates. The purinebases (Pu) include adenine(A), guanine(G) and derivatives and analogsthereof. The pyrimidine bases (Py) include cytosine (C), thymine (T),uracil (U) and derivatives and analogs thereof. Examples of suchderivatives or analogs, by way of illustration and not limitation, arethose which are modified with a reporter group, biotinylated, aminemodified, radiolabeled, alkylated, and the like and also includephosphorothioate, phosphite, ring atom modified derivatives, and thelike. The reporter group can be a fluorescent group such as fluorescein,a chemiluminescent group such as luminol, a terbium chelator such asN-(hydroxyethyl) ethylenediaminetriacetic acid that is capable ofdetection by delayed fluorescence, and the like.

The term sdNTP represents a thio deoxynucleotide wherein N=G, A, T or C.

The term bdNTP represents a borano deoxynucleotide wherein N=G, A, T, orC.

The term bdPuTP represents a borano-substituted purine.

The term bdPyTP represents a borano-substituted pyrimidine.

The term dPu represents a deoxypurine.

The term dPy represents a deoxypyrimidine.

The procedures disclosed herein which involve the molecular manipulationof nucleic acids are known to those skilled in the art. See generallyFredrick M. Ausubel et al. (1995), “Short Protocols in MolecularBiology,” John Wiley and Sons, and Joseph Sambrook et al. (1989),“Molecular Cloning, A Laboratory Manual,” second ed., Cold Spring HarborLaboratory Press as incorporated herein by reference.

Accordingly, the present invention provides materials, processes andkits for directionally ligating a nucleic acid to an adaptor sequence.The processes and kits utilize an. amplification mixture including apolymerase, a deoxynucleotide triphosphate (dNTP) mixture comprisingmodified dNTPs, a first primer and a second primer. A portion of thefirst and second primers are complimentary to the first and secondstrand of the double stranded target nucleic acid, respectively. Thefirst primer has a first terminus which is complimentary to a firstligation site sequence of the first adaptor sequence and the secondprimer has a second terminus which is complimentary to a second ligationsite sequence of a second adaptor sequence. The primers are extendedusing the dNTP mixture containing the modified dNTPs, which during theextension step of the PCR, are incorporated into the amplificationproduct in lieu of one of the non-modified dNTPs. Amplicons are producedwhich contain the modified and non-modified dNTPs. The amplicons have afirst and second terminus which are complimentary to the first ligationsite sequence of a first adaptor sequence and the second ligation sitesequence of a second adaptor sequence, respectively. The amplicons arecontacted with an exonuclease which enzymatically degrades the ampliconby removing the non-modified nucleotides at the termini of the ampliconsthereby exposing the sequences complimentary to either the firstligation site sequence of the first adaptor sequence and the secondligation site sequence of the second adaptor sequence. The firstterminus of the digested amplicon is then ligated to the first ligationsite sequence of the first adaptor sequence, and preferably, the secondterminus of the digested amplicon is also ligated to the second ligationsite sequence of the second adaptor sequence.

The present invention relates to processes for directionally ligating anucleic acid to an adaptor sequence which utilize an amplificationmixture to amplify a target nucleic acid thereby producing an amplifiedproduct, or an amplicon, from the target nucleic acid sequence.Preferably, the amplification mixture comprises a polymerase; a dNTPmixture comprising modified dNTPs for at least one of the fournucleotide triphosphates, i.e., dATP, dGTP, dCTP, dTTP and analogsthereof; a first primer; and a second primer. The amplicon is digestedwith an exonuclease and ligated to a first adaptor sequence. In oneembodiment, the adaptor sequence is a cloning vector. Accordingly, oneaspect of the present invention is directed to processes for cloning anucleic acid into a cloning vector.

The target nucleic acid is amplified using methods of amplificationknown in the art. Any nucleic acid specimen can be utilized as thestarting nucleic acid template. Thus, the amplification process mayemploy DNA or RNA, wherein DNA or RNA may be double or single stranded.In the event that RNA is to be used as a template, enzymes, and/orconditions optimal for reverse transcribing the template to DNA known tothose in the art would be utilized. Preferably, the target nucleic acidis a double stranded DNA.

Several in vitro amplification techniques may be modified using methodsknown in the art, to use a DNA polymerase to enzymatically synthesize aDNA from the target nucleic acid, e.g., synthesis of RNA from DNA bytranscription is followed by reverse transcription. Several suitabletechniques include but are not limited to, the polymerase chain reaction(PCR) method, transcription-based amplification system (TAS),self-sustained sequence replication system (3SR), ligation amplicationreaction (LAR), Qβ RNA replication system and run-off transcription. Apreferred method of amplification is PCR amplification which involves anenzymatic chain reaction in which exponential quantities of the targetnucleic acid are produced relative to the number of reaction stepsperformed. PCR amplification techniques and many variations of the PCRare known and well documented. See e.g., Saiki et al., Science 239:487-491 (1988); U.S. Pat. Nos. 4,682,195, 4,683,202 and 4,800,159, whichare incorporated herein by reference.

Typically, the selected nucleic acid, preferably, double stranded DNA,is denatured, thus forming single strands which are used as templates.One oligonucleotide primer is substantially complimentary to thenegative (−) strand and another primer is substantially complimentary tothe positive (+) strand. DNA primers are DNA sequences capable ofinitiating synthesis of a primer extension product. Primers“substantially complimentary” to each strand of the target nucleic acidsequence will hybridize to their respective nucleic acid strands underfavorable conditions known to one skilled in the art, e.g., pH, saltconcentration, cation concentration, temperature.

The primers used herein are complimentary to the first and second strandof the target nucleic acid to be amplified. The first primer contains afirst terminus complimentary to a portion of the first strand of thetarget nucleic acid and complimentary to a first ligation site sequenceof the first adaptor sequence. The second primer contains a secondterminus complimentary to a portion of the second strand of the targetnucleic acid and complimentary to a second ligation site sequence of thesecond adaptor sequence. The sequences of the first and second ligationsite sequences of the first and second adaptor sequence are notidentical and therefore, the sequences complimentary to the first andsecond ligation site sequences are not identical. It is not requiredthat the sequence complimentary to a portion of the target nucleic acidand the sequence complimentary to the ligation site sequence of theadaptor sequence be exclusive of one another. Accordingly, two primersare utilized wherein the first terminus of the first primer iscomplimentary to a first ligation site sequence of the first adaptorsequence and the second terminus of the second primer is complimentaryto a second ligation site sequence in the second adaptor sequence.

The first terminus of the first primer can be either a 3′ terminus or a5′ terminus. Likewise, the second terminus of the second primer can beeither a 3′ terminus or a 5′ terminus. In a preferred embodiment, thefirst terminus of the first primer is a 5′ terminus which iscomplimentary to the first ligation site sequence in a first adaptorsequence and the second terminus of the second primer is a 5′ terminuswhich is complimentary to the second ligation site sequence in thesecond adaptor sequence, wherein the 5′ termini of the first and secondprimers are not identical.

It is preferred that the first primer's terminus which is complimentaryto the first ligation site sequence of the first adaptor sequence beapproximately between one and ten nucleotides in length, more preferablytwo to seven nucleotides in length, still more preferably two to fivenucleotides in length, and most preferably four nucleotides in length.It is also preferred that the second primer's terminus which iscomplimentary to the second ligation site sequence of the second adaptorsequence be approximately between one to ten nucleotides in length, morepreferably two to seven nucleotides in length, still more preferably twoto five nucleotides in length, and most preferably four nucleotides inlength. In a preferred embodiment, the 5′ terminus of the first primercontains a four nucleotide sequence complimentary to a first ligationsite sequence in the first adaptor sequence, and the 5′ terminus of thesecond primer contains a four nucleotide sequence complimentary to asecond ligation site sequence in the second adaptor sequence, whereinthe 5′ terminus of the first primer is not identical to the 5′ terminusof the second primer.

Several methods are known in the art which are used to produce 5′overhang sequences. For example, 5′ overhang sequences can be producedby a modification of the so called “exchange or replacement labeling”reaction. See Sambrook et al., “Molecular Cloning”, Second Ed., page5.39. This method utilizes a 3′ to 5′ exonuclease bearing DNA polymerase(typically T4 DNA polymerase) to burn back to the first occurrence of asingle nucleotide included in the reaction mix.

In a preferred embodiment, the 3′ or 5′ termini of the first and secondprimers which are complimentary to the first and second ligation sitesequences of the first and second adaptor sequence, respectively, arerestriction enzyme recognition sequences. Many restriction enzymes areknown in the art, such as those available from New England Biolabs,Beverly, Mass. Preferably, the 5′ termini of the first and secondprimers contain restriction enzyme recognition sites corresponding to arestriction enzyme which leaves a 5′ overhang restriction site. The 5′termini of the first and second primers contain restriction enzymerecognition sites which are not identical to each other. While 5′overhang restriction sites are preferred, the first and second primerstermini containing restriction enzyme recognition sites corresponding toa restriction enzyme which leaves a 3′ overhang restriction site mayalso be used.

In a preferred embodiment, the 5′ terminus of the first primer sequenceis an Acc65I, AflII, AgeI, AcaI, ApoI, AvrII, BamHI, BglII, BsiWI, EagI,EcoRI, HindIII, NcoI, NgoMIV, NheI, NotI, SalI, XbaI, XhoI or XmaIrecognition sequence and the 3′ terminus of the second primer sequenceis an Acc65I, AflII, AgeI, AcaI, ApoI, AvrII, BamHI, BglII, BsiWI, EagI,EcoRI, HindIII, NcoI, NgoMIV, NheI, NotI, SalI, XbaI, XhoI or XmaIrecognition sequence. Preferably, the sequences of the 5′ termini of thefirst and second primer are not identical.

Annealing the primers to the denatured DNA templates is followed byextension with an enzyme to result in newly synthesized + and − strandscontaining the target DNA sequence. This annealing process consists ofthe hybridization of the primer to complimentary nucleotides of the DNAsequence template in a buffered aqueous solution. It is understood thatthe nucleotide sequence of the primer need not be completelycomplimentary to the portion of the DNA template in order to effectivelyanneal to the DNA template. The mixture containing the DNA templates isthen heated to a temperature sufficient to separate the twocomplimentary strands of DNA. In a preferred embodiment, the mixturecontaining the DNA templates is heated to about 90 to 100° C. from about0.5 to 10 minutes, even more preferably from 0.5 to 4 minutes to allowthe DNA templates to denature and form single strands. The reaction mixis next cooled to a temperature sufficient to allow the primers tospecifically anneal to sequences flanking the gene or sequence ofinterest. Preferably, the reaction mixture is cooled to 50 to 60° C.,for approximately 0.5 to 5 minutes.

Preferably, the enzyme catalyzing the extension reaction is apolymerase, and more preferably, the polymerase used to amplify thetarget nucleic acid is a thermostable polymerase. Heat-stable(thermophilic) DNA polymerases are particularly preferred as they arestable when PCR is conducted in a single solution in which thetemperature is cycled. Representative heat-stable polymerases are theDNA polymerases isolated from Bacillus stearothermophilus (Bio-Rad,Richmond, Calif.), Thermus thermophilus (FINZYME, ATCC #27634), Thermusspecies (ATCC #31674), Thermus aquaticus strain TV 1151B (ATCC #25105)and Thermus filiformis (ATCC #43280), the polymerase isolated fromThermus flavus (Molecular Biology Resources; Milwaukee, Wis.).Particularly preferred is Taq DNA polymerase available from a variety ofsources including PerkinElmer, (Boston, Mass.), Promega (Madison, Wis.)and Stratagene (La Jolla, Calif.), and AmpliTaq™ DNA polymerase, arecombinant Taq DNA polymerase available from PerkinElmer.

The polymerase extends the primer by incorporating dNTPs from the dNTPmixture into the resulting polynucleotide, or amplicon. Preferredpolymerases incorporate the modified dNTPs efficiently,base-specifically and independent of the surrounding sequence context.In a preferred embodiment, the polymerase used to amplify the targetnucleic acid is Taq, REDTaq, AccuTaqLA, AmpliTaq™, KlenTaqLA, Ultma,Pwo, or Pfu and more preferably, Taq polymerase, REDTaq, AccuTaqLA andAmpliTaq™. A combination of two or more of the above polymerases suchas, for example, a combination of Taq and Pfu, may also be used in theprocesses of the present invention. The temperature of the reactionmixture is then set to the optimum for the DNA polymerase to allow DNAextension to proceed.

The modified and unmodified dNTPs in the dNTP mixture are incorporatedinto the resulting polynucleotide, or amplicon. For use in a process ofdirectionally ligating a nucleic acid to an adaptor sequence, the dNTPmixture comprises modified dNTPs for at least one of the four nucleotidetriphosphates comprising dATP, dGTP, dCTP, dTTP and analogs thereof. Inone preferred embodiment, the adaptor sequence is a cloning vector andthe dNTP mixture for use in a process of cloning a nucleic acid into acloning vector comprises modified dNTPs for at least two of the fournucleotide triphosphates comprising dATP, dGTP, dCTP, dTTP and analogsthereof. It will be appreciated that by virtue of the present inventionthat all modified dNTPs which serve as a substrate for a polymerase andprotect nucleotides and/or the phosphodiester linkages between thenucleotides from enzymatic degradation by an exonuclease can be utilizedin the dNTP mixture of the present invention. Preferably, any molecularmanipulation, e.g., modifications to the base, sugar and/or phosphate,which produces a modified dNTP which serves as a substrate for a DNApolymerase and protects nucleotides and/or the phosphodiester linkagesbetween the nucleotides from enzymatic degradation by an exonuclease canbe utilized in the dNTP mixture of the present invention.

Preferably, the polymerase incorporates the modified dNTPs efficiently,base-specifically and independent of the surrounding sequence context.It is also preferred that the modified dNTPs in the dNTP mixture do notinhibit the enzyme catalyzed incorporation of the modified andunmodified dNTPs into the nucleotide sequence of the amplicon.Preferably, the modified dNTPs minimally affect the efficiency of theincorporation of the modified and unmodified dNTPs. In a preferredembodiment, the modified dNTPs reduce the efficiency of the polymeraseincorporation of modified and unmodified dNTPs into the polynucleotidesequence of the amplicon by approximately 60%, more preferably, byapproximately 70%, even more preferably by approximately 80%, and mostpreferably by approximately 90%. In a preferred embodiment, the modifieddNTPs reduce the efficiency of the polymerase incorporation of modifiedand unmodified dNTPs into the polynucleotide sequence of the amplicon byless than 90%.

Preferably, the dNTP mixture comprises modified dNTPs wherein eachmodified dNTP is a fraction of the total amount of the particulardeoxynucleotide triphosphate base in the dNTP mixture. In a process fordirectionally ligating a nucleic acid to a first adaptor sequence, thedNTP mixture contains modified dNTPs for at least one of the four dNTPscomprising dATP, dGTP, dCTP, dTTP and analogs thereof, preferably, thedNTP mixture contains modified dNTPs for at least two of th four dNTPscomprising dATP, dGTP, dCTP, dTTP and analogs thereof. dNTP₁ representsthe at least one modified dNTP in the dNTP mixture, if two modifieddNTPs are present in the dNTP mixture, dNTP₂ represents the second ofthe at least two modified dNTPs in the dNTP mixture, if three modifieddNTPs are present in the dNTP mixture, dNTP₃ represents the third of theat least two modified dNTPs in the dNTP mixture, and if four modifieddNTPs are present in the dNTP mixture, dNTP₄ represents the fourth ofthe at least two modified dNTPs in the dNTP mixture. Preferably, thedNTP mixture comprises modified dNTPs for at least two dNTPs, modifieddNTP₁ and modified dNTP₂. Each modified dNTP₁ and modified dNTP₂ can bea modified dATP, modified dGTP, modified dCTP or modified dTTP, providedthat dNTP₁ and dNTP₂ are not the same modified dNTP. The modified dNTPsare enzymatically incorporated into the amplicons in lieu of one of thenon-modified dNTPs during primer extension. The modified dNTPs impartresistance by blocking the action of the exonuclease, for example,exonuclease III, or any other 3′ to 5′ exonuclease that cleaves normalphosphodiester linkages between the nucleotides but to which themodified dNTPs prove resistant.

In one embodiment, the dNTP mixture comprises three modified dNTPs,dNTP₁, dNTP₂ and dNTP₃, wherein each modified dNTP is a fraction of thetotal amount of the particular deoxynucleotide triphosphate base in thedNTP mixture. Each modified dNTP₁, dNTP₂ and dNTP₃ can be a modifieddATP, modified dGTP, modified dCTP or modified dTTP, provided thatdNTP₁, dNTP₂ and dNTP₃ are not the same modified dNTP. In anotherembodiment, the dNTP mixture comprises four modified dNTPs, dNTP₁,dNTP₂, dNTP₃ and dNTP₄, wherein each modified dNTP is a fraction of thetotal amount of the particular deoxynucleotide triphosphate base in thedNTP mixture. Each modified dNTP₁, dNTP₂, dNTP₃ and dNTP₄ can be amodified dATP, modified dGTP, modified dCTP or modified dTTP, providedthat modified dNTP₁, dNTP₂, dNTP₃ and dNTP₄ are not the same modifieddNTP. The modified dNTPs are enzymatically incorporated into theamplicons in lieu of one of the non-modified dNTPs during primerextension. The modified dNTPs impart resistance by blocking the actionof the exonuclease, for example, exonuclease III, or any other 3′ to 5′exonuclease that cleaves normal phosphodiester linkages between thenucleotides but to which the modified dNTP proves resistant.

In a process for directionally ligating a nucleic acid to a firstadaptor sequence, the dNTP mixture contains modified dNTPs for at leastone of the four dNTPs comprising dATP, dGTP, dCTP and dTTP. dNTP₁represents the at least one modified dNTP in the dNTP mixture usedspecifically for directional ligation, and dNTP₁ can be a modified dATP,modified dGTP, modified dCTP or modified dTTP. In a preferredembodiment, the dNTP mixture utilized for directionally ligating anucleic acid to an adaptor sequence comprises two modified dNTPs,modified dNTP₁ and dNTP₂. The modified dNTPs in the dNTP mixture areenzymatically incorporated into the amplicons in lieu of one of thenon-modified dNTPs during primer extension. The modified dNTPs impartresistance by blocking the action of the exonuclease, for example,exonuclease III, or any other 3′ to 5′ exonuclease that cleaves normalphosphodiester linkages between the nucleotides but to which themodified dNTP proves resistant.

The dNTP mixture utilized in a process for directionally ligating anucleic acid to an adaptor sequence comprises at least one modified dNTPcomprising a modified dATP, modified dGTP, modified dCTP or a modifieddTTP. Preferably, the at least one modified dNTP is a fraction of thetotal amount of the particular deoxynucleotide triphosphate base in thedNTP mixture. Preferably, the modified dNTPs are modified purines orpyrimidines, more preferably, the modified dNTPs are alpha phosphatemodified purines or pyrimidines, even more preferably, alpha phosphatemodified dATP or dGTP, and most preferably, alpha phosphate modifieddGTP. In a preferred embodiment, the dNTP mixture utilized fordirectional ligation comprises at least one modified dNTP wherein themodified dNTPs are alpha boranophosphorano dNTPs or alphathiophosphorano dNTPs, more preferably, the modified dNTPs are alphaboranophosphorano dNTPs. In another preferred embodiment, the modifieddNTPs in the dNTP mixture utilized for directional ligation are alphaboranophosphorano dATPs, alpha boranophosphorano dGTPs, alphathiophosphorano dATPs or alpha thiophosphorano dGTPs, more preferably,the modified dNTPs in the dNTP mixture are alpha boranophosphoranodATPs, alpha boranophosphorano dGTPs, and most preferably, the modifieddNTPs are alpha boranophosphorano dGTPs.

Preferred nucleotide boranotriphosphates, e.g.,5′-alpha-boranotriphosphates and methods of synthesis are disclosed inU.S. Pat. Nos. 5,260,427, 5,659,027 and 5,683,869, and preferrednucleotide thiotriphosphates, e.g., 5′-alpha-thiotriphosphates andmethods of synthesis are disclosed in Labeit et al., Meth. Enzymol.1987, 155: 166 and Nakamaye et al., Nucl. Acids. Res., 1988, 16: 9947,the entirety of which are incorporated herein by reference. Onceincorporated into the polynucleotide, thiophosphorodiester andboranophosphodiester bonds are resistant to exonuclease digestion.

Preferably, the dNTP mixture utilized to directionally ligate a nucleicacid to a first adaptor sequence comprises modified dNTPs for at leasttwo of the four dNTPs comprising dATP, dGTP, dCTP and dTTP which arepreferably modified purines, modified pyrimidines, or a combination ofmodified purines and pyrimidines. More preferably, the modified dNTPs inthe dNTP mixture comprise alpha phosphate modified dNTPs, still morepreferably, the modified dNTPs in the dNTP mixture comprise alphaphosphate modified purines, alpha phosphate modified pyrimidines or acombination of alpha phosphate modified purines and alpha phosphatemodified pyrimidines, and most preferably, alpha phosphate modified dATPand dGTP. In a preferred embodiment, the modified dNTPs in the dNTPmixture comprise a combination of alpha boranophosphorano dNTPs andalpha thiophosphorano dNTPs, more preferably, the modified dNTPs in thedNTP mixture comprise alpha thiophosphorano dNTPs, and most preferably,the modified dNTPs in the dNTP mixture comprise alpha boranophosphoranodNTPs. In another preferred embodiment, the modified dNTPs in the dNTPmixture comprise a combination of alpha boranophosphorano dATP and dGTPand alpha thiophosphorano dATP and dGTP, more preferably, the modifieddNTPs in the dNTP mixture comprise alpha thiophosphorano dATP and dGTP,and most preferably, the modified dNTPs in the dNTP mixture comprisealpha boranophosphorano dATP and dGTP.

The amount of the four nucleotide triphosphates in the dNTP mixture isdetermined by the concentration of modified dNTPs relative to theconcentration of non-modified dNTPs. In a process for directionallyligating a nucleic acid to a first adaptor sequence, the dNTP mixturecomprises modified dNTPs for at least one of four dNTPs comprising dATP,dCTP, dGTP and dTTP. Preferably, the amount of the four nucleotidetriphosphates in the dNTP mixture utilized for directional cloning isdetermined by the ratio (R₁) of the concentration of modified dNTP₁([modified dNTP₁]) relative to the concentration of non-modified dNTP₁([dNTP₁]) which is shown as follows:R ₁=[modified dNTP₁]/[dNTP₁]In a preferred embodiment, the ratio of the concentration of modifieddNTP₁ relative to the concentration of non-modified dNTP₁ is less than20, more preferably, the ratio is less than 9, and even more preferably,the ratio is less than 1.

Preferably, modified dNTP₁ in the dNTP mixture utilized in a process fordirectionally cloning a nucleic acid into a vector are modified purines,and more preferably, alpha phosphate substituted purines. In a preferredembodiment, the ratio of the concentration of alpha phosphatesubstituted dNTP₁ relative to the concentration of non-modified dNTP₁ isabout 0.05 to 20, more preferably, the ratio is about 0.05 to 10, andmost preferably, 0.05 to 4.

In a preferred embodiment, modified dNTP₁ is alpha thiophosphorano dATP,alpha thiophosphorano dGTP, alpha boranophosphorano dATP or alphaboranophosphorano dGTP, more preferably, dNTP₁ is alphaboranophosphorano dATP or alpha boranophosphorano dGTP, and mostpreferably, dNTP₁ is alpha boranophosphorano dGTP.

In a preferred embodiment, dNTP₁ in the dNTP mixture utilized todirectionally ligate a nucleic acid to a first adaptor sequence arealpha thiophosphorano dNTPs, more preferably, alpha thiophosphorano dATPor dGTP and most preferably, alpha thiophosphorano dGTP. In onepreferred embodiment, modified dNTP₁ is alpha thiophosphorano dATP andthe ratio of the concentration of alpha thiophosphorano dATP relative tothe concentration of non-modified dATP is 0.05 to 10, more preferably,1.0 to 9, and most preferably, 1.5 to 5. More preferably, modified dNTP,is alpha thiophosphorano dGTP and the ratio of the concentration ofalpha thiophosphorano dGTP relative to the concentration of non-modifieddGTP is 0.05 to 10, more preferably, 0.05 to 7, and most preferably, 1.0to 3.0.

In a more preferred embodiment, dNTP, in the dNTP mixture utilized todirectionally ligate a nucleic acid to an adaptor sequence are alphaboranophosphorano dNTPs, more preferably, alpha boranophosphorano dATPor dGTP and most preferably, alpha boranophosphorano dGTP. Preferably,modified dNTP₁ is alpha boranophosphorano dATP and the ratio of theconcentration of alpha boranophosphorano dATP relative to theconcentration of non-modified dATP is 0.05 to 10, more preferably, 0.05to 4, and most preferably, 0.01 to 2.0. In a more preferred embodiment,modified dNTP₁ is alpha boranophosphorano dGTP and the ratio of theconcentration of alpha boranophosphorano dGTP relative to theconcentration of non-modified dGTP is 0.05 to 20, more preferably, 0.1to 10, and most preferably, 0.1 to 5.

In a preferred embodiment, the dNTP mixture comprises modified dNTPs fortwo of the four dNTPs, modified dNTP₁ and modified dNTP₂. Preferably,the amount of the four nucleotide triphosphates in the dNTP mixture isdetermined by the ratio (R₂) of the concentration of modified dNTP₁([modified dNTP₁]) to the concentration of modified dNTP₂ ([modifieddNTP₂]) relative to the concentration of non-modified dNTP₁ ([dNTP₁]) tothe concentration of non-modified dNTP₂ ([dNTP₂]) which is shown asfollows:R ₂=([modified dNTP₁]/[modified dNTP₂])/([dNTP₁]/[dNTP₂])In a preferred embodiment, the ratio of the concentration of modifieddNTP₁ to the concentration of modified dNTP₂ relative to theconcentration of non-modified dNTP, to the concentration of non-modifieddNTP₂ is less than 51, more preferably, the ratio is less than 27, andeven more preferably, the ratio is less than 13.

Preferably, the modified dNTP, and modified dNTP₂ in the dNTP mixtureare modified purines and more preferably, alpha phosphate substituteddNTPs. In a preferred embodiment, the ratio of the concentration ofalpha phosphate substituted dNTP₁ to the concentration of alphaphosphate substituted dNTP₂ relative to the concentration ofnon-modified dNTP₁ to the concentration of non-modified dNTP₂ is about0.05 to 6.4, more preferably, the ratio is about 0.1 to 3.2, even morepreferably, the ratio is about 0.2 to 1.6.

In a preferred embodiment, the two modified dNTPs in the dNTP mixtureare alpha thiophosphorano dNTPs, and more preferably, alphathiophosphorano dGTP and alpha thiophosphorano dATP . Preferably, theratio of the concentration of alpha thiophosphorano dGTP to theconcentration of alpha thiophosphorano dATP relative to theconcentration of non-modified dGTP to the concentration of non-modifieddATP is between 0.8 to 5.3, more preferably, the ratio is between 0.17to 2.7, even more preferably, the ratio is between 0.33 to 1.33, andmost preferably, the ratio is about 0.66.

In another preferred embodiment, the modified dNTPs in the dNTP mixtureare alpha boranophosphorano dNTPs, and more preferably, alphaboranophosphorano dGTP and alpha boranophosphorano dATP . Preferably,the ratio of the concentration of alpha boranophosphorano dGTP to theconcentration alpha boranophosphorano dATP relative to the concentrationof non-modified dGTP to the concentration of non-modified dATP isbetween 0.05 to 6.4, more preferably, the ratio is between 0.1 to 3.2,even more preferably, the ratio is between 0.2 to 1.6, and mostpreferably, the ratio is about 0.4.

In another embodiment, the dNTP mixture comprises three modified dNTPs,dNTP₁, dNTP₂ and dNTP₃ wherein each modified dNTP is a fraction of thetotal amount of the particular deoxynucleotide triphosphate base in thedNTP mixture. Each dNTP₁, dNTP₂ and dNTP₃ is a modified dATP, dCTP, dGTPor dTTP, provided that dNTP₁, dNTP₂ and dNTP₃ are not the same modifieddNTP. The amount of the four nucleotide triphosphates in the dNTPmixture comprising three modified dNTPs is determined by the ratio (R₃)of the product of the concentration of modified dNTP₁ ([modifieddNTP₁]), the concentration of modified dNTP₂ ([modified dNTP₂]) and theconcentration of modified dNTP₃ ([modified dNTP₃]) relative to theproduct of the concentration of non-modified dNTP₁ ([dNTP₁]), theconcentration of non-modified dNTP₂ ([dNTP₂]) and the concentration ofnon-modified dNTP₃ ([dNTP₃]), which is shown as follows:R₃ = ([modified  dNTP₁] × [modified  dNTP₂]×  [modified  dNTP₃])/([dNTP₁] × [dNTP₂] × [dNTP₃])

In order to determine the concentration of each modified dNTP₁, dNTP₂,and dNTP₃ in the dNTP mixture, an acceptable ratio of each modified andnon-modified dNTP₁, dNTP₂, and dNTP₃ is individually determined bytitration. An acceptable ratio is obtained when a compromise betweenamplification toxicity is minimized and protection against exonucleasedigestion is maximized. In a preferred embodiment, this is when PCRamplification yield and quality is identical to reactions performed inthe absence of any modified nucleotides while exonuclease protection isessentially quantitative, i.e., exonuclease digestion can result in amean overhang length of less than 20 base pairs. It is possible howeverthat a reduction in amplification yield may need to be tolerated so thatadequate protection can be obtained. The type of experiment isexemplified in the s-dNTP and b-dNTP titrations shown in FIGS. 6 and 9.See “Quality Improvement through Planned Experimentation 2nd Edition”,Ronald Moen, Thomas Nolan, Lloyd Provost, McGraw Hill, N.Y., 1998,incorporated herein by reference.

After the preferred concentration of each individual modified andnon-modified dNTP₁, dNTP₂, and dNTP₃ is determined, the total mixture ofmodified and non-modified dNTP₁, dNTP₂, and dNTP₃ is determined bytitration. The composition of the dNTP mixture containing modified dNTPsfor three of the four dNTPs are obtained in a fashion analogous to thatused for the determination of the relative ratios of solutionscontaining two modified dNTPs as described in Examples 3 and 4 andexemplified in FIG. 7. However, to determine the relative ratios ofmodified to non-modified dNTPs in a dNTP mixture comprising modifieddNTPs for three of the four dNTPs, a three dimensional titration ratherthan two dimensional titration would be performed.

In another embodiment, the dNTP mixture comprises four modified dNTPs,dNTP₁, dNTP₂, dNTP₃ and dNTP₄ wherein each modified dNTP is a fractionof the total amount of the particular deoxynucleotide triphosphate basein the dNTP mixture. Each dNTP₁, dNTP₂, dNTP₃ and dNTP₄ is a modifieddATP, dCTP, dGTP or dTTP, provided that dNTP₁, dNTP₂, dNTP₃ and dNTP₄are not the same modified dNTP. The amount of the four nucleotidetriphosphates in the dNTP mixture comprising four modified dNTPs isdetermined by the ratio (R₄) of the product of the concentration ofmodified dNTP₁ ([modified dNTP₁]), the concentration of modified dNTP₂([modified dNTP₂]), the concentration of modified dNTP₃ ([modifieddNTP₃]) and the concentration of modified dNTP₄ ([modified dNTP₄])relative to the product of the concentration of non-modified dNTP₁([dNTP₁]), the concentration of non-modified dNTP₂ ([dNTP₂]), theconcentration of non-modified dNTP₃ ([dNTP₃]) and the concentration ofnon-modified dNTP₄ ([dNTP₄]), which is shown as follows:R ₄=([modified dNTP₁]×[modified dNTP₂]×[modified dNTP₃]×[modifieddNTP₄])/( [dNTP₁]×[dNTP₂]×[dNTP₃]×[dNTP₄])

In order to determine the concentration of each modified dNTP₁, dNTP₂,dNTP₃, and dNTP₄ in the dNTP mixture, an acceptable ratio of eachmodified and non-modified dNTP₁, dNTP₂, dNTP₃, and dNTP₄ is individuallydetermined by titration. An acceptable ratio of each modified tonon-modified dNTP is obtained when a compromise between PCRamplification toxicity is minimized and exonuclease protection ismaximized. In a preferred embodiment, an acceptable ratio is obtainedwhen PCR amplification yield and quality is identical to reactionsperformed in the absence of any modified dNTP in the dNTP mixture whileexonuclease protection is essentially quantitative, i.e., exonucleasedigestion results in a mean overhang length of less than 20 base pairs.However, sacrifices in amplification yield may need to be tolerated sothat adequate exonuclease protection can be obtained. The type ofexperiment is exemplified in the s-dNTP and b-dNTP titrations shown inFIGS. 6 and 9. After the preferred ratio of each individual modified andnon-modified dNTP₁, dNTP₂, dNTP₃, and dNTP₄ is determined, the totalcomposition of modified and non-modified dNTP₁, dNTP₂, dNTP₃, and dNTP₄in the dNTP mixture is determined by titration. The method for a dNTPmixture comprising four modified dNTPs are obtained using methodsanalogous to that utilized for the determination of the relative ratiosof solutions containing two or three modified nucleotides as describedin Examples 3 and 4 and exemplified in FIG. 7. A four dimensionaltitration rather than a two or three dimensional titration would beperformed to determine the relative ratios of modified to non-modifieddNTPs in a dNTP mixture comprising modified dNTPs for four dNTPs. See“Quality Improvement through Planned Experimentation 2nd Edition”,Ronald Moen, Thomas Nolan, Lloyd Provost, McGraw Hill, N.Y., 1998,incorporated herein by reference.

An amplicon is produced from the extension of the primers when dNTPs,both modified and unmodified dNTPs, in the dNTP mixture areenzymatically incorporated into the nucleotide sequence of the amplicon.Thus, the newly synthesized amplicon contains both modified andunmodified dNTPs. The incorporation of the modified dNTPs impartresistance against enzymatic degradation by an exonuclease at the siteof incorporation of the modified dNTPs thereby protecting thenucleotides of the amplicon from enzymatic degradation by anexonuclease.

The extension of the first and second primers using the dNTP mixtureresult in newly synthesized amplicons containing both modified andunmodified dNTPs which have a first terminus complimentary to a firstligation site sequence of the first adaptor sequence and a secondterminus complimentary to a second ligation site sequence of a secondadaptor sequence. Preferably, the amplicon's termini are amplifiedsequences which correspond to the 3′ or 5′ termini of the first andsecond primer sequences used in PCR. In a preferred embodiment, thefirst ligation site sequence of the first adaptor sequence and thesecond ligation site sequence of the second adaptor sequence are theends of a cloning vector. Preferably, the termini of the amplicon arecomplimentary to the first and second ligation site sequences of thecloning vector, and more preferably, the 5′ termini of the ampliconscontain sequences complimentary to the first and second ligation sitesequences of the cloning vector. In an even more preferred embodiment,the 5′ termini of the amplicons contain restriction enzyme recognitionsequences. Preferably, the termini of the amplicon contain restrictionenzyme recognition sequences corresponding to restriction enzymes whichleave a 5′ overhang restriction site. In a preferred embodiment, thetermini of the amplicon are not identical to each other to enabledirectional ligation into the cloning vector.

In order to prepare amplicons which can be inserted in one directionrelative to the first adaptor sequence, the dNTP mixture comprisingmodified dNTPs for at least one, and preferably, two of the four dNTPscomprising dATP , dGTP, dCTP and dTTP is utilized to extend first andsecond primers which contain a first and second sequence, respectively,which is complimentary to a first and second ligation site sequence ofthe adaptor sequence, respectively. In a preferred embodiment, the firstligation site sequence of the first adaptor sequence and the secondligation site sequence of the second adaptor sequence are the ends of acloning vector and the amplicons are prepared for directional cloninginto the cloning vector.

The extension of the first and second primers using the dNTP mixturecomprising modified dNTPs for at least one, and preferably, two of thefour dNTPs result in newly synthesized amplicons containing bothmodified and unmodified dNTPs. The amplicons have a first terminuscomplimentary to a first ligation site sequence of the first adaptorsequence and a second terminus complimentary to a second ligation sitesequence of the second adaptor sequence wherein the termini of theamplicon are not identical to each other. Preferably, the amplicon'stermini are amplified sequences which correspond to the 3′ or 5′ terminiof the first and second primer sequences used in PCR. In a preferredembodiment, the first and second ligation site sequences of the firstand second adaptor sequences, respectively, are the ends of a cloningvector. Preferably, the termini of the amplicon are complimentary to thefirst and second ligation site sequences of the cloning vector, and morepreferably, the 5′ termini of the amplicons contain sequencescomplimentary to the first and second ligation site sequences of thecloning vector wherein the first and second ligation site sequences ofthe cloning vector are not identical. In an even more preferredembodiment, the 5′ termini of the amplicons contain restriction enzymerecognition sequences. Preferably, the termini of the amplicon containrestriction enzyme recognition sequences corresponding to restrictionenzymes which leave a 5′ overhang restriction site.

The amplicons produced from this process have a variety of uses. Forexample, RNA transcripts are synthesized from the amplicons by in vitroDNA transcription. Transcription of RNA is performed with theappropriate RNA polymerase, preferably, T3, T7, or SP6, depending on theRNA polymerase promoter sites present upstream of the DNA to betranscribed. Because these polymerases are extremely promoter-specific,i.e., there is almost no transcriptional cross-talk, virtuallyhomogenous RNA can be obtained using plasmid DNA (circular orlinearized) or enzymatically synthesized DNA (linear) as the template ina transcription reaction. Melton et al. (Nucleic Acid Research (1984) v.12, p. 7035) describes a general procedure for RNA synthesis in vitro.In short, DNA is mixed with an RNA polymerase plus the fourribonucleoside triphosphates used in RNA synthesis. During an incubationat 37° C., large amounts of the desired RNA are then generated by invitro transcription. RNA transcripts may be radiolabeled with ³²P-,³³P-, ³⁵S-, or ³H-labeled ribonucleotides, depending upon the specificapplication.

RNA transcripts may be used to generate probes for hybridization toNorthern or Southern blots, plaque and colony lifts, tissue sections andchromosome spreads. RNA transcripts are also useful for Si nucleasemapping, generation of antisense RNAs to block translation, and mRNAsynthesis for translation in vitro.

Additionally, the amplicons may be used to synthesize protein expressionin vitro. The in vitro synthesis of proteins in cell-free extracts is animportant tool for molecular biologists and has a variety ofapplications, including the rapid identification of gene products,localization of mutations through synthesis of truncated gene products,protein folding studies, and incorporation of modified or unnaturalamino acids for functional studies. The use of in vitro translationsystems can have advantages over in vivo gene expression when theover-expressed product is toxic to the host cell, when the product isinsoluble or forms inclusion bodies, or when the protein undergoes rapidproteolytic degradation by intracellular proteases.

The most frequently used cell-free translation systems consist ofextracts from rabbit reticulocytes, wheat germ and Escherichia coli. Allare prepared as crude extracts containing all the macromolecularcomponents (70S or 80S ribosomes, tRNAs, aminoacyl-tRNA synthetases,initiation, elongation and termination factors, etc.) required fortranslation of exogenous RNA. Pelham et al. (Eur. J. Biochem. (1976) v.67, p. 247) describe a general procedure for mRNA translation in vitro.In short, the lysates containing the cellular components necessary forprotein synthesis are mixed with the subject mRNA and incubated at 30°C. for 90 minutes followed by an analysis of the translation reactionfor expected products (i.e., SDS-PAGE analysis).

The amplicons containing modified dNTPs and unmodified dNTPs arepreferably purified and treated with an exonuclease. Preferably, theamplicons were synthesized using a dNTP mixture comprising modifieddNTPs for at least two of the four nucleotide triphosphates comprisingdATP, dGTP, dCTP and dTTP and analogs thereof. Even more preferably, theamplicons were synthesized using a dNTP mixture comprising modified dATPand dGTP, and still more preferably, alpha thiophosphorano dATPs anddGTPs or alpha boranophosphorano dATPs and dGTPs, and most preferably,alpha boranophosphorano dATPs and dGTPs. Accordingly, in a preferredembodiment, the amplicons comprise alpha phosphate modified dATPs anddGTPs.

Because the modified dNTPs impart resistance to enzymatic degradation byblocking the action of an exonuclease that cleaves normal phosphodiesterbonds, the exonuclease will only digest unmodified dNTPs which have beenincorporated into the nucleotide sequence of the amplicon. Preferably,the enzymatic degradation by an exonuclease at the sites ofincorporation of the modified dNTPs is approximately 1.1 to 10 timesslower than exonuclease degradation of unmodified phosphodiester bondhydrolysis, more preferably, the enzymatic degradation by an exonucleaseat the sites of incorporation of the modified dNTPs is approximately 10to 1000 times slower than exonuclease degradation of unmodifiedphosphodiester bond hydrolysis, even more preferably the enzymaticdegradation by an exonuclease at the sites of incorporation of themodified dNTPs is approximately 1000 to 10,000 times slower thanexonuclease degradation of unmodified phosphodiester bond hydrolysis,and most preferably, the enzymatic degradation by an exonuclease at thesites of incorporation of the modified dNTPs is approximately 10,000 to100,000 slower than exonuclease degradation of unmodified phosphodiesterbond hydrolysis. In a preferred embodiment, the enzymatic degradation byan exonuclease at the sites of incorporation of the modified dNTPs iscompletely blocked at each modified phosphodiester bond of the amplicon.The amplicons are protected from enzymatic degradation by theexonuclease at the sites of incorporation of the modified dNTPs andpreferably, the termini of the digested amplicons terminate at the sitesof incorporation of the modified dNTPs. The exonuclease digests thetermini of the amplicons thereby creating a single stranded overhangsequence at each terminus of the amplicon.

Preferably, the single stranded overhang sequence at the first terminusof the amplicon is complimentary to a first ligation site sequence ofthe first adaptor sequence, and the single stranded overhang sequence atthe second terminus of the amplicon is complimentary to the secondligation site sequence of the second adaptor sequence. The sequences ofthe first and second ligation site sequences of the first adaptorsequence and the second adaptor sequence are not identical therebyenabling for the ligation of the digested amplicon in one directionrelative to the first and second adaptor sequences. Preferably, thefirst and second ligation site sequences of the first and second adaptorsequences, respectively, are the ends of a doubly digested cloningvector and the digested amplicon is preferably inserted into the cloningvector in one direction relative to the vector, i.e., the digestedamplicon is directionally cloned into the vector.

The amplicon is treated with an exonuclease, wherein the exonuclease canbe a 5′ to 3′ exonuclease or a 3′ to 5′ exonuclease. In a preferredembodiment, the amplicons are treated with a 3′ to 5′ exonuclease, andmore preferably, the amplicons are treated with exonuclease III.Treating the amplicons with exonuclease III digests the nucleotides ofthe 3′ termini and exposes single stranded overhang sequences at each 5′terminus of the amplicons. Preferably, the single stranded overhangsequence at the first 5′ terminus of the digested amplicon is similar,and preferably identical to the first primer's terminus which iscomplimentary to a first ligation site sequence of the first adaptorsequence; and the single stranded overhang sequence at the second 5′terminus of the digested amplicon is similar, and preferably identicalto the second primer's terminus which is complimentary to a secondligation site sequence of the second adaptor sequence.

In a preferred embodiment, the single stranded overhang sequences at thefirst and second 5′ termini of the digested amplicon are complimentaryto the first and second ligation site sequences in the first and secondadaptor sequence, respectively. Preferably, the single-stranded overhangsequence at the first and second 5′ terminus is approximately betweenone and ten nucleotides in length, more preferably two to sevennucleotides in length, still more preferably two to five nucleotides inlength, and most preferably four nucleotides in length. In a preferredembodiment, the single overhang sequence at the first and second 5′terminus of the digested amplicon has similar, and more preferably, thesame number of nucleotides as the first and second ligation sitesequence of the first and second adaptor sequences, respectively, andthe single overhang sequence at the first and second 5′ terminus of thedigested amplicon is complimentary to the first and second ligation sitesequence of the first and second adaptor sequences, respectively.

The single overhang sequence at the first 5′ terminus of the digestedamplicon is directionally ligated to the first adaptor sequence to forman amplicon-adaptor complex. Preferably, the adaptor sequence is alinear synthetic or enzymatically prepared linear nucleic acid. In apreferred embodiment, the single overhang sequence at the first andsecond 5′ terminus of the digested amplicon is directionally ligated tothe first and second ligation site sequence of the first and secondadaptor sequences, respectively, thereby producing a digested ampliconflanked by a first and second adaptor sequence. In a preferredembodiment, the digested amplicon flanked by the first and secondadaptor sequence is subjected to a second round of PCR amplificationusing primers which are specific for the first and second adaptors.

In a preferred embodiment, RNA is synthesized from the amplicon flankedby the first and second adaptor sequences by in vitro transcription. SeeMelton et al., Nucleic Acid Res., 1984, 12: 7035. The synthesized RNAmay be used to generate probes for hybridization to Northern or Southernblots, plaque and colony lifts, tissue sections and chromosome spreads.RNA transcripts are also useful for S1 nuclease mapping, generation ofantisense RNAs to block translation, and mRNA synthesis for translationin vitro. In another preferred embodiment, the amplicon flanked by thefirst and second adaptor sequences is used to synthesize protein by invitro translation. See Pelham et al., Eur. J. Biochem., 1976, 67: 247.

In one preferred embodiment, the first and second ligation sitesequences of the first and second adaptor sequences are the ends of acloning vehicle such as a plasmid, vector, or bacterial artificialchromosome. These cloning vehicles may be used for subcloning and/orgene expression. Aside from being used to propagate the digestedamplicon, the adaptor sequence may be designed to impart one or moredesired properties that add functionality to the adaptor sequence.

Preferably, the adaptor sequence comprises a nucleotide sequenceencoding for expression systems which will permit affinity purification.In a preferred embodiment, the adaptor sequence may comprise at leastone epitope tag which may be c-myc, polyhistidine, polyarginine,glutathione-S-transferase (GST) tag, HA epitope, V5, Xpress™, and FLAG®.See Evan et al., Mol Cell Biol. 5:3610-3616 (1985). The use of apolyarginine tag allows for the polypeptide to be purified on a cationexchange resin. See Sassenfeld, H. M. and Brewer, S. J. BioTechnology,2:76 (1984); U.S. Pat. No. 4,532,207. Preferably, the amplicon-adaptorcomplex includes a nucleotide sequence encoding forglutathione-S-transferase. The resulting polypeptide may be selectivelyrecovered on glutathione-agarose. See Smith, D. B. and Johnson, K. S.Gene 67:31 (1988). In another preferred embodiment, the adaptor sequencecomprises a nucleotide sequence encoding IgG-Sepharose can be used toaffinity purify fusion proteins containing staphylococcal protein A. SeeUhlen, M. et al. Gene 23:369 (1983). In yet another preferredembodiment, the adaptor sequence comprises a nucleotide sequenceencoding the maltose-binding protein domain from the malE gene of E.coli which allows the affinity purification of the resulting polypeptideon amylose resins.

In a preferred embodiment, the adaptor sequence is designed to containsequences encoding for a metal chelating sequence composed of multipleor alternating histidine residues which would allow the adaptor sequenceto bind to a metal ion immobilized on a resin or other matrix.Preferably, a metal chelating sequence may comprise at least onehistidine residue, at least one glycine residue or a combination ofalternating or multiple histidine residues, which may be used inaffinity purification techniques using a Ni²⁺ binding metal resin. Seee.g., U.S. Pat. Nos. 4,569,794, 5,310,663, 5,284,933 and 5,594,115 whichare incorporated herein by reference. Once the polypeptide is bound tothe metal resin, the adaptor sequence can be released by protonation ofits associated metal ion-binding ligand. Dissociation is achieved bylowering the pH of the surrounding buffer medium, a common method knownin the art for eluting bound proteins.

In another preferred embodiment, the adaptor sequence comprises nucleicacid sequences encoding for an epitope tag. Epitope tagging utilizesantibodies against guest peptides to study protein localization at thecellular level and subcellular levels. See Kolodziej, P. A. and Young,R. A., Methods Enzymol., 194:508-519 (1991). Preferably, the digestedamplicon is ligated to the first adaptor sequence comprising anucleotide sequence encoding the epitope tag to form an amplicon-adaptorcomplex. The amplicon-adaptor complex is introduced into a cell by amethod such as transformation. When the amplicon-adaptor complex isexpressed the result is a chimeric protein containing the epitope as aguest peptide. If the epitope is exposed on the surface of the protein,it is available for recognition by the epitope-specific antibody,allowing the investigator to observe the protein within the cell usingimmunofluorescence or other immunolocalization techniques. Preferably,the amplicon-adaptor complex comprising such epitope tags are used forpurifying proteins utilizing affinity purification techniques.

Preferably, the nucleotide sequence encoding the epitope tag is locatedat the terminus of the adaptor sequence. After the digested amplicon isdirectionally ligated to the adaptor sequence to form anamplicon-adaptor complex, the amplicon-adaptor complex is expressed.Preferably, the nucleotide sequence encoding the epitope tag is locatedat the terminus of the amplicon-adaptor complex, and more preferably,the nucleotide sequence encoding the epitope tag is located at theamino-terminus of the amplicon-adaptor complex. Generally, when theepitope is fused to the amino or carboxy-terminus of the expressedprotein, the epitope is more accessible to the antibody for detectionand less likely to cause severe structural or functional perturbations.

In a preferred embodiment, the adaptor sequence comprises a nucleotidesequence encoding for the FLAG® octapeptideAsp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys (SEQ ID NO: 10). Preferably, the FLAG®epitope is fused to the amino or carboxy-terminus of the expressedamplicon-adaptor complex protein, and more preferably, the FLAG® epitopeis fused to the amino terminus of the expressed amplicon-adaptor complexprotein. The placement of FLAG® octapeptide at the amino-terminus allowsfor the amplicon-adaptor complex to be affinity purified on animmuno-affinity resin containing an antibody specific for theoctapeptide. See Hopp, T. P., et al. Biotechnology, 6:1204 (1988);Prickett, K. S., et al., BioTechniques, 7:580 (1989); and U.S. Pat. No.4,851,341. The original FLAG® sequence is recognized by two antibodies,M1, M2, and a FLAG® sequence with an initiator methionine attached isrecognized by a third antibody, M5. The last five amino acids of theFLAG® sequence is a recognition site for the protease enterokinase,thus, allowing for removal of the FLAG® epitope. More preferably, theadaptor sequence comprises a nucleotide sequence encoding for multipleantigenic epitopes, and even more preferably, the adaptor sequencecomprises a nucleotide sequence encoding for multiple FLAG® epitopes.See U.S. patent application Ser. No. 09/415,000, filed Oct. 8, 1999,incorporated herein.

The adaptor sequence may also comprise other elements such as promoters,repressors, or enhancers, e.g., BNL, CMV, T7, T3, SP6, Gal4, Tet On/Off.Preferably, the adaptor sequence comprises a reporting element, e.g.,fluorescing proteins (e.g. GFP etc.), beta galactosidase (β-gal),luciferase, and/or an antibiotic resistant gene, (e.g. amp, neo, agr,kan, pur, hyg and etc.). The usage of a reporting gene allows for cloneselection after the amplicon-adaptor complex is expressed. Othernucleotide sequences which may be included in the adapter nucleic acidare sequences that direct recombination or enable ligase independentcloning. The adaptor sequence may also be designed to include sequencescritical to developing nucleic acid based diagnostics, increasedspecificity diagnostics, SNP analysis or gene expression analysis.

In one preferred embodiment, the digested amplicon is ligated to acloning vehicle, and more preferably, a cloning vector. Preferably, thefirst and second ligation site sequences of the first and second adaptorsequences, respectively, are the ends of a cloning vector therebyresulting in a cloning vector comprising a ligation site flanked by afirst and second ligation site sequence to which the digested ampliconmay be ligated. The first and second ligation site sequences arecomplimentary to the single stranded overhang sequences at the first andsecond terminus of the digested amplicon, respectively, wherein thenucleotide sequences of the first and second ligation site sequences arenot identical. The difference in the “sticky ends” or cohesive ends ofeach digested amplicon's termini and the complementarity between thefirst and second terminus of the digested amplicon and the first andsecond ligation site sequences, respectively, allow for the digestedamplicons to be directionally inserted into the cloning vector.

Preferably, the synthesis of amplicons using a dNTP mixture comprisingmodified dNTPs followed by exonuclease digestion results in a higherpreponderance of directional clones. In a preferred embodiment, a highpercentage of the digested amplicons are inserted directionally into thecloning vector. Preferably, 80% of the digested amplicons are inserteddirectionally into the cloning vector, more preferably, 90% of thedigested amplicons are inserted directionally into the cloning vector,and most preferably, 99% of the digested amplicons are inserteddirectionally into the cloning vector.

The resulting cloning vector is used to transform a host microorganism.The transformants are isolated and analyzed for the presence of thetarget nucleic acid. The transformants are then multiplied in culture tocause replication of the vector. Various procedures and materials forpreparing recombinant vectors, transforming host cells with the vectors,replicating the vector and expressing polypeptide and proteins arediscussed by Old and Primrose, Principles of Gene Manipulation, (2d Ed.1981).

The digested amplicon may be inserted into any of a variety ofconventional cloning vectors. Although plasmids are preferred, thevector may be alternatively a bacteriophage or cosmid. If cloning takesplace in mammalian or plant cells, viruses can be used as vectors. If aplasmid is employed, it may be obtained from a natural source orartificially synthesized. The particular plasmid chosen should becompatible with the particular cells serving as the host, whether abacteria such as Escherichia coli (E. coli), yeast, or other unicellularmicroorganism. The plasmid should have the proper origin of replication(replicon) for the particular host cell chosen. Any variety of cell thatis transformable may serve as a host cell. Examples include but are notlimited to, E. coli XL1-blue, DH5-α, HB101, JM101, JM103, JM109, etc.Other bacterial hosts may include Bacillus or Pseudomonas species andthe like. By way of example, eukaryotic host cells may includeSaccharomyces species.

This invention also contemplates kits for directional ligation of anucleic acid to an adaptor sequence. This invention further contemplateskits for cloning of a nucleic acid to an adaptor sequence. Such kits mayinclude, for example, the dNTP mixture, instructions for using the dNTPmixture and other components necessary for directional ligation or forcloning. The kit may be in the form of a test kit, that is, in apackaged collection or combination as appropriate for the needs of theuser and any analytical instrumentation involved. Minimally, the kitwill comprise the dNTP mixture comprising modified dNTPs which, whenincorporated into a polynucleotide, impart resistance against enzymaticdigestion by an exonuclease at the sites of incorporation of themodified dNTPs. Preferably, the kit furthers contain an exonuclease,preferably a 3′ to 5′ exonuclease, and more preferably, exonuclease III.The kit can, of course, also include appropriate packaging, containers,labeling, buffers, and controls for directional ligation of a nucleicacid to an adaptor sequence or for cloning a nucleic acid into a cloningvector.

The following examples are included to demonstrate preferred embodimentsof the invention. It should be appreciated by those of skill in the artthat the techniques disclosed in the examples that follow representtechniques discovered by the inventors to function well in the practiceof the invention. However, those of skill in the art should, in light ofthe present disclosure, appreciate that many changes can be made in thespecific embodiments that are disclosed and still obtain a like orsimilar result without departing from the spirit and scope of theinvention, therefore all matter set forth or shown in the accompanyingdrawings is to be interpreted as illustrative and not in a limitingsense.

EXAMPLES Example 1 In vitro Incorporation of Thiodeoxypurines into DNAusing Polymerases

The universality with respect to thermostable polymerase was checkedusing a variety of commercially available polymerases. The experimentwas performed with respect to product quality and quantity for a normal(0.8 mM dNTP) vs. thiodeoxypurine containing dNTP (sdNTP) mix. Theexperiment was carried out using buffers and reagents with the exceptionof the nucleotide mix as described or supplied by the enzyme supplier.Taq, REDTaq, AccuTaqLA, KlenTaqLA were obtained from Sigma-Aldrich, St.Louis, Mo. Pfu Turbo was obtained from Startagene. Vent and Deep Ventwere obtained from New England Biolabs. Pwo was obtained from Roche.UlTma was obtained from Perkin Elmer. Amplification products wereanalyzed on a 0.2 agarose gel and visualized by ethidium bromidestaining. No attempt was made to optimize cycling parameters for anyparticular enzyme.

FIG. 2 demonstrates that product quality was independent of nucleotidemix as the products from a normal vs. osdNTP were identically composed.Vent/Deep Vent lack of product was not followed up since neither enzymeis licensed for PCR and each have a reputation for being problematic.Notably from the gel, all enzymes seem to have somewhat of a reducedyield using the sdNTP mix formulation.

FIG. 3 summarizes the amplification and protection yields from a ³²pdNTP containing PCR. Yields were measured by TCA precipitation. The toppanel shows overall yields of amplification (incorporation) products forall enzymes normalized to Taq/dNTP. Yields were generally within 2× ofTaq. The middle panel shows yields of sdNTP products normalized to dNTP.The inclusion of thiodeoxypurines resulted in a reduction of ampliconyield for all enzymes tested. The reduction is likely inconsequentialfor Taq, REDTaq, AmpliTaqLA, KlenTaq and Ultma.

Example 2 Stability of Phosphothioate Bonds to Hydrolysis by ExonucleaseIII

This experiment demonstrates that thiodeoxypurines incorporated into theamplicons demonstrated resistance to Exonuclease III hydrolysis. Thelower panel of FIG. 3 demonstrates that while all products were degradedfrom approximately 50% or less (+exo/−exo) for the all deoxynucleotideamplifications, the addition of ExoIII to the thiodeoxypurine containingreactions resulted in protections ranging from approximately 80 to 250%.

Protection of nucleotides greater than 100% may occur if the polymeraseis active enough during ExoIII degradation to incorporate additionallabel. This was demonstrated by performing PCR in the absence of alabeled radionucleotide which was followed immediately by addition ofthe radionucleotide as part of the ExoIII addition (FIG. 4). Using thesdNTP mix, all enzymes incorporated nucleotides significantly abovebackground.

FIG. 5 demonstrates that ExoIII and polymerase were not competitivelyadding/removing nucleotides to produce an abundance of blunt ends. PCRwas performed in the absence of labeled dNTP followed by addition ofExoIII/³²PdCTP. Taq DNA polymerase was added to half of the samples andfollowed by incubation at 72° C. to completely fill in the ExoIIIgenerated 5′ overhangs. As shown, re-incorporation during ExoIIIdigestion replaced an insignificant portion (approximately 5-15%) of thenucleotides removed by ExoIII. These data demonstrated that it is safeto use the sdNTP nucleotide mix with most thermostable DNA polymerases.

Example 3 Optimization of Incorporation of Thiodeoxynucleotides andExonuclease Digestion

The shortcoming of using a single nucleotide for controlling exoIIIdigestion is sequence specific over and under-digestion. Forover-digestion it is conceivable that target sequences could beamplified that are under-represented by the exonuclease controllingnucleotide (e.g. low abundance dG sequences have a low occurrence of sdGoccupation). In this event, over-digestion of the amplicon would resultin a population of clonable duplex DNAs and single stranded-ligationcompeting sequences. Such a situation would undoubtedly result indiminished cloning efficiencies relative to single strand deficientduplex amplicons. This is most likely going to be problematic forrelatively short and/or high AT (low GC) amplicons. Under-digestionresults from incomplete exposure of the cohesive 5′ ends upon exoIIIdigestion leading to reduced cloning efficiency from sub-optimal insert:plasmid stoicheometry and from competitive inhibitory duplexes. Usingone modified nucleotide (sdGTP) produced amplicons whose sdG content was30%. There was a 30% chance that every G position would be occupied by athioG. Exonuclease III then has a 30% chance of stopping at each G.Palindromic restriction sites can contain between one and two dGsopposite dC of a 5′ overhang. Table 1 contains the expected proportionsof A, B and C after exoIII digestion of amplicons designed to be clonedbetween dG containing and non-dG containing restriction sites (one endprobabilities) and between two dG contining sites (both ends). The mainlesson from this exercise is that the concentration of competitive ends(column C) approaches and exceeds the concentration of desired amplicon(column A). With the exception of the both end-two dG containingrestriction examples (Table 1 column B bold), amplicons B are of littleconcern. TABLE 1 Reduction of ligation efficiencies due to 30% dGTPaSoccupation. Fraction of digested amplicon^(a) One end^(b) Both ends^(c)Recognition A B C A B C sequence^(d) Affected enzymes^(e) 0.7 0 0.3 0.490.09 0.42 {circumflex over ( )}CDDD Bsp11I, AflIII, NcoI, StyI, DsaI,BspHI, SpeI, AvrII, StyI, NheI, XbaI {circumflex over ( )}DCDD XhoI,AvaI, BsoBI, SmlI {circumflex over ( )}DDCD HindIII, SfcI, ApaLI{circumflex over ( )}DDDC DpnII, MboI, Sau3AI, BglII, BstYI, BamHI,BclI, BsiWI, Acc65I, BanI, BsrGI N{circumflex over ( )}CGN MaeII, AclI,BsaHI, MspI, HpaII, HinP1I, NarI, TaqI, ClaI, BspDI, XhoI, AvaI, BsoBI,SmlI, AccI, BstBI N{circumflex over ( )}GCN None 0.49 0 0.51 0.24 0.260.50 {circumflex over ( )}CCGG AgeI, BsrFI, BsaWI, XmaI, AvaI, BsoBI,NgoMIV, BsrFI, BspEI, BsaWI {circumflex over ( )}CGCG MluI, AflIII,DsaI, BssHII {circumflex over ( )}GCGC KasI, BanI {circumflex over( )}GGCC EagI, EaeI, Bsp120Ibased upon 30% dGTPaS occupation.

Amplicon to be cloned into plasmid digested with non-dG overhang (e.g.EcoRI) and dG containing overhang (e.g. affected enzyme column).

Amplicon to be cloned into plasmid digested with two dG containingoverhang restrioction enzymes (e.g. one or two enzymes from affectedenzyme column).

D=not C, {circumflex over ( )}=cut position

Palindromic Tetra- and hexa-nucleotide recognition sequences table (NewEngland Biolabs)

Having established that phosphorothioate bonds can provide protectionfrom the hydrolytic action of exonuclease III, this experiment wasundertaken to understand the possibility of formulating a PCR invisiblenucleotide mixture that would produce uniformly gapped ligation productsindependent of sequence would have nuclease resistant phosphodiestersstatistically represented at all positions. With phosphorothioates, thiscould be accomplished by including all four sdNTPs. The final totalnucleotide concentration used in this formulation was 0.8 mM, aconcentration that is commonly recommended in many PCR protocols. Atthis concentration, the free Mg⁺⁺ concentration should not changerelative to a “normal” or typical PCR. It should be noted that the 0.8mM concentration is only required if, as here, it is desired that thereaction formulation of the reaction buffer is to be ignored. It ispossible that other nucleotide formulations could be investigated,however, it would be prudent to ascertain their effect on free magnesiumconcentration and adjust accordingly as PCR performance is oftendependent on magnesium concentration.

Sequence independent gap size or probability would occur when allnucleotides are replaced by odNTPS at identical frequency. Suchformulation would meet the following criteria:0.8  mM = [dATP] + [dCTP] + [dGTP] + [dTTP] + [sdATP]+  [sdCTP] + [sdGTP] + [sdTTP] andk_(As)[sdATP]=k_(Cs)[sdCTP=k_(Gs)[sdGTP]=k_(Ts)[sdTTP]wherein k_(Ns)=incorporation rate constant.

The possibility of formulating the above solution was to titrate PCRs(lambda 500 mer) with each thionucleotide. PCR was performed usinglambda control primers (Perkin Elmer) and lambda DNA (Sigma) astemplate. All reagents (1× PCR buffer, 0.05 u/μl Taq DNA polymerase)with the exception of the nucleotide composition were from Sigma andused without modification. Cycling conditions were 94° C./30 sec., 68°C./30 sec., 72° C./60 sec. for 30 cycles.

PCR was performed using an invariant ³²PDN′TP tracer and 0.2mM=[dNTP]+[sdNTP] (N and N′ are not identical). Thionucleotideincorporation was ascertained by resistance to ExoIII digestion. Asshown in the top panel of FIG. 6, PCR is tolerant of thionucleotides inthe order G>A>T≈C. The lower panel of FIG. 6 shows that the ampliconswere preferentially protected from exoIII digestion in the orderG>A>T>C. PCR yield data indicated that the concentration of any of thedNTPs should remain above 0.1 mM in order to minimally impact PCR yield.This allowed for 0.4 mM to be available for sdNTP.

Supposing that each thionucleotide was equally distributed (i.e.[sdNTP]=0.1 mM each), then the concentrations of sdNTP would besub-optimal for protection. Instead, thiopurines were chosen foroptimization since they are best utilized by Taq during amplification.Although this relaxed the requirement for sequence independent ExoIIIdigestion termination, it still expanded the repertoire of sequencesrelative to the single thionucleotide example. The formulation criteriathen became:0.8 mM=[dATP]+[dCTP]+[dGTP]+[dTTP]+[sdATP]+[sdGTP]andk_(As)[sdATP]=k_(Gs)[sdGTP].

From the above thionucleotide titration yield data, the concentration ofeach dNTP was greater than or equal to 0.1 mM. PCR yields for asdATP/sdGTP titration at [dNTP]=0.125 mM each (N=A,C,G,T) were from ca.85-92% (0.3 mM sdATP to 0.3 mM sdGTP) and PCR yields were 59-72% at[dNTP]=0.1 mM each. 100% was defined at [dNTP]=0.2 mM each for theseexperiments. From this data, an acceptable formulation would be:[dNTP]=0.125 mM each and 0.3 mM=[sdATP]+[sdGTP].

The optimal ratio of sdATP to sdGTP (i.e. such thatk_(As)[sdATP]=k_(Gs)[sdGTP]) was measured as shown in FIG. 7. Althoughthese conditions were not those used in the final formulation, thequantity measured is the ratio of thiodeoxypurines. Inspection of thefigure revealed that k_(As)[sdATP]=k_(Gs)[sdGTP] at 0.3 mM sdATP/0.2 mMsdGTP or [sdATP]/[sdGTP]=3/2. The formulation was finalized by titratingthiodeoxypurine (sdATP/sdGTP=3/2) vs. deoxypurine (dATP/dGTP=1). FIG. 8(top panel) demonstrates that PCR product yield begins to suffer atconcentrations of thiodeoxypurine above 0.3 mM. The final formulationwas: [dNTP]=0.125 mM each, 0.18 mM [sdATP] and 0.12 mM [sdGTP]. Thelower panel of FIG. 8 reports the occupation of purine sites by dPuMP asa function of dPuTP concentration. The above formulation producesamplicons that are 97% dPuMP/3% sdPuMP (i.e. dAMP occupation was 97% at[dPuTP]=0.25 mM). The expected 5′ overhang length for a random sequenceduplex is calculated as (0.5+0.5*0.97)^(n) (i.e. 50% chance dPy plus 50%chance of 97% chance dPu at nucleotide position n). From this, it isapparent that all but very short amplicons should remain duplex afterExoIII digestion (data not shown).

In random sequence DNA, this nucleotide solution will produce ampliconsthat are 50% blocked to exoIII digestion by base 43. The probability ofencountering a dG deficient sequence decrease as 0.75^(n) compared to adPu deficient sequence at 0.5^(n) i.e., a dG deficient sequence is1.5^(n) more likely than is a dPu deficient sequence. Table 2demonstrates how quickly this difference manifests itself as a functionof base position. The advantage of sdPu incorporation vs. sdG is givenby:(P_(ntg)P_(N))/(P_(notPu)P′_(N))^(n)where P_(notG) is the probabilty of not G (i.e. is A, C or T), P_(N) isprobability of dNMP (A+C+G+T), P_(notPu) is probability of not A or G(i.e. C+T), P′_(oN) is probability of dNMP (A+C+G+T) or:(0.75(0.75+0.25P_(oG)))/(0.5 (0.5+0.5P_(oPu)))^(n)

where P_(oG) and P_(oPu) are the probabilities of an dGMP (70%) anddPuMP (97%) at base position n (column 3, Table 2). These datademonstrate that though specific base occupation has been reduced usingsdPuTP, the effect is superior protection for a larger cross section ofsequences. TABLE 2 Relative probabilities of encountering dG vs dPudeficient sequences as a function base position dG vs. dPu deficiencysdPu vs. sdG advantage Base probabilities (0.75(0.75 + 0.25 × 0.7))/Position (0.75/0.5)^(n) (0.5(0.5 + 0.5 × 0.97))^(n) 1 1.5 1.4 5 7.59 5.610 57.7 31 15 438 170 20 3,325 960 25 25,251 5,400 30 191,751 30,000 351,456,109 160,000 40 11,057,332 930,000 45 83,966,617 5,200,000 50637,621,500 29,000,000 55 >human genome 1.6 × 10⁸

Table 3 contains expected proportions of “ligatable” ends from sdPuTPcontaining PCR products. TABLE 3 Reduction of ligation efficiencies dueto 6% sdPuTP occupation Fraction of digested amplicon^(a) One end^(b)Both ends^(c) A B C A B C Recognition sequence^(d) N/A N/A N/A 0.880.003 0.11 {circumflex over ( )}PuPuPyPy, {circumflex over ( )}PuPyPuPy,{circumflex over ( )}PyPuPyPu, {circumflex over ( )}PyPyPuPu N/A N/A N/A0.94 0.0009 0.058 N{circumflex over ( )}PyPuN, N{circumflex over( )}PuPyN

Calculated using 3% sdPuTP occupation.

All four base palindromes contain purines making it impossible to havean unaffected end.

Amplicon to be cloned into plasmid digested with one or two four baseoverhang producing restriction enzymes.

Pu=dA or dG, Py=dC or dT, {circumflex over ( )}=cut position

A comparison of Tables 1 and 3 reveals that inclusion of an additionalthionucleotide reduces the relative abundance of competitive ligationinhibitors to inconsequential levels. However, a nucleotide mixturecontaining a set of three or four modified nucleotides could bedeveloped that would produce low enough occupancies at any positionwithin the amplicon thereby retaining a minimization of competitiveligation inhibitors.

Although there was a loss in product yield using the osdNTP mix, the mixdid not alter product quality relative to PCR using non-modified dNTPmixtures (data not shown).

Example 4 Optimization of Incorporation of bdNTPs and ExonucleaseDigestion

The methods used to develop a phosphoroborano containing deoxynucleotidemix that rendered amplicons protected from exoIII overdigestion wasanalogous with the thiophosphate work described in Example 3.Specifically, 1) titration of all four phosphoroboranes in aquantitative PCR to establish nucleotide biases, 2) optimization offavored phosphoroborane ratios (i.e. bdATP, bdGTP), 3) titration ofoptimized phosphoroborane mixture with dNTPs. PCR was performed usinglambda control primers (Perkin Elmer) and lambda DNA (Sigma) astemplate. All reagents (1× PCR buffer, 0.05 u/μl Taq DNA polymerase)with the exception of the nucleotide composition were from Sigma andused without modification. Cycling conditions were 94° C./30 sec., 68°C./30 sec., 72OC/60 sec for 30 cycles.

FIG. 9 contains titration results for the four phosphoroboranes (bdNTP,N=A, C, G, T). These data show that PCR yields are negatively impactedby phosphoroboranes in the order G<A<C<T and that ExoIII protectionincreases in the order G>A>C≈T. These results indicate that bdPyTPs wereincorporated inefficiently and the insensitivity of PCR yield to bdPyTPconcentration may be largely a result of dPyTP dilution, i.e., thatbdPyTPs are not particularly inhibitory. As with the thionucleotides, itis evident that Taq polymerase prefers bdPuTPs and that in keeping witha 0.8 mM total nucleotide triphosphate concentration, there would againbe little room to include all bdNTPs in a reaction mix. A reasonablecompromise again would be to formulate a mix that contains dNTP+bdPuTPat a total of 0.8 mM. As with the thiophosphates, this is not arequirement if one is willing to investigate the affect other nucleotideformulations have on free magnesium concentration and hence PCRperformance. FIGS. 10 and 11 contain bdATP/bdGTP and dPuTP/bdPuTPoptimization results respectively. PCR was performed using lambdacontrol primers (Perkin Elmer) and lambda DNA (Sigma) as template. Allreagents (1× PCR buffer, 0.05 u/μl Taq DNA polymerase) with theexception of the nucleotide composition were from Sigma and used withoutmodification. Cycling conditions were 94° C./30 sec., 68° C./30 sec.,72° C./60 sec for 30 cycles. The optimal bdATP/bdGTP ratio is 2.5 andverified by the overlapping data in the lower panel of FIG. 11. Theuseful (high protection levels) and un-impeding (little or no effect onPCR yield) concentrations of bdPuTP range from approximately 0.1 to 0.3mM bdPuTP. The mean 5′ overhangs at these concentrations are 81 and 4bases respectively. From these data it is clear that a nucleotide mixcould be formulated using bdNTPs.

Example 5 Directional Cloning of a PCR Product into a Vector

The present example is provided to demonstrate the exonuclease recessiontechnique for providing directional cloning of a PCR product into avector. The clonability of amplicons prepared using the above describednucleotide formulations and ExoIII digestion were compared withamplicons cloned using restriction enzyme generation of the cohesiveends. Until this point, amplification had been indiscriminatelyperformed using Taq or REDTaq DNA polymerases. Accutaq LA and REDTaqwere used for the below experiments. For integrity, Exoclone vs.restriction enzyme cloning was compared. Thus, the lambda 500mer wasamplified using primer set BamHI 1 (SEQ ID NO: 1) and XbaI 1 (SEQ ID NO:2) for Exoclone, and a primer set designed for digestion with BamHI/XbaI(BamHI 2: cut BamHI 1 lam 5′ GCACG GGATCC GAT GAG TTC GTG TCC GTA CAACTG (SEQ ID NO: 3), XbaI 2: cut XbaI 1 lam 5′ GCACG TCTAGA GGT TAT CGAAAT CAG CCA CAG CGC (SEQ ID NO: 4), recognition site underlined). AfterPCR the amplicons were respectively digested with ExoIII, BamHI/XbaI,ligated with BamHI/XbaI cut pUC19 and transformed into competent E. coliDH5a (Life Technologies). PCR was performed using above describedprimers and lambda DNA (Sigma) as template. All reagents (1× PCR buffer,0.05 u/μl Taq DNA polymerase) with the exception of the nucleotidecomposition were from Sigma and used without modification. Cyclingconditions were 94° C./30 sec., 68° C./30 sec., 72° C./60 sec for 30cycles. Restriction digestion was performed using successive digestionsby the restriction enzymes using a silica bind and elute DNApurification method (Qiagen PCR purification kit) between and afterdigestions. Ligation was at 16° C. for two hours using reagents from aligation kit (Sigma).

The plasmids were isolated and subjected to PCR across the inserts.Amplification across the insert (primer set EcopUC (SEQ ID NO:5)/HindpUC (SEQ ID NO: 6), FIG. 12) yielded amplicons that were largerthan from pUC19 and the same as an amplification product from apreviously prepared construct containing an identical insert (FIG. 13Alanes 1-4 vs. 5 and 6 respectively). FIG. 13B demonstrates that all ofthe clones were directional. That is, primer pairs EcopUC (SEQ ID NO:5)/Lambda R (SEQ ID NO: 8) and HindpUC (SEQ ID NO: 6)/Lambda L (SEQ IDNO: 7) produced no PCR products while primer pairs EcopUC (SEQ ID NO:5)/Lambda L (SEQ ID NO: 7) and HindpUC (SEQ ID NO: 6)/Lambda R (SEQ IDNO: 8) produced PCR products. For additional evidence, one of the REclones (pBX) was cut using BamHI, XbaI and BamHI/XbaI (FIG. 14). Thedouble digest yielded the expected “insert” restriction fragment (FIG.14 lane 4). The singly cut plasmid had a higher electrophoretic mobilitythan did singly cut pUC19 (compare FIG. 14 lanes 1, 3 vs. 4, 5). Removalof the insert by double digestion resulted in a fragment that comigratedwith cut pUC19 (lane 4 vs. 5-7).

Example 6 Phosphoborano Modified Nucleotides vs. PhosphorothioateModified Nucleotides

This experiment was conducted in order to determine whether anydifference in cloning exists between the use of a mixture containingphosphoborano modified nucleotides and a mixture containingphosphorothioate modified nucleotides. FIG. 15 demonstrates the expectedoverhang length as a function of modified nucleotide concentration.Clearly, phosphoborano nucleotides can be used at higher occupationrates with out impacting PCR performance.

Applicants then determined whether there was some advantage to ligatingDNAs with shorter rather than longer 5′ overhangs. For this PCRreactions (template=lambda DNA, primer set=BamHI 1 (SEQ ID NO: 1)/XbaI 1(SEQ ID NO: 2)) at two bdPuTP and one sdPuTP (FIG. 15) concentrationwere ligated after exoIII digestion to BamHI/XbaI cut pUC19. PCR wasperformed using above described primers and lambda DNA (Sigma) astemplate. All reagents (1× PCR buffer, 0.05 u/μl Taq DNA polymerase)with the exception of the nucleotide composition were from Sigma andused without modification. Cycling conditions were 94° C./30 sec., 68°C./30 sec., 72° C./60 sec for 30 cycles. Ligation reactions wereserially diluted 10× to obtain relative ligation efficiencies from thePCRs. FIG. 16 shows resulting PCR products. Ligation was at 16° C. fortwo hours using reagents from a ligation kit (Sigma). Lanes marked bxwere parallel PCR performed with an authentic clone. All ligationreactions produced a relatively high molecular weight PCR product whileonly EcopUC/lambda L and HindpUC/lambda R produced products thatco-migrated with the authentic clone amplification products. The highmolecular weight products are likely amplification of dimerized insert.Relative ligation efficiencies were estimated by quantifying the bandsfrom FIG. 16 using a BioRad gel doc image analysis system.

Example 7 Preponderance or Lack Thereof of Mutations at Single StrandOverhangs

Transformations using plasmids that potentially contain relatively longtracts of single strand sequence is non-typical. In all likelihood,cellular polymerases would repair the single stranded “lesion” makingthe inserted DNA entirely duplex. It is however not inconceivable thatcellular repair enzymes could react unexpectedly and introduce unwantedmutations in the “lesion” region.

To investigate the cellular response fidelity, bacterial AlkalinePhosphatase (SEQ ID NO: 8) was PCR amplified from a gene bearing plasmidand cloned in E. coli (Nova Blue). Amplification was performed using thethionucleotide mix described in Example 3. 96 clones were sequencedusing fluorescent cycle sequencing kit (Applied Biosystems) and analyzedfor mutations as a function of mutation position vs. duplex probability.The results are shown in Table 4. TABLE 4 Base position and fractionduplex at mutation sites Base Position Fraction Duplex Number ofmutations 23 0.579477 1 24 0.579477 1 29 0.604708 1 34 0.65072 1 550.82316 1 137 0.987638 2 158 0.993342 1 198 0.997814 1 207 0.998068 1209 0.998184 4 214 0.998492 2 215 0.998582 1 234 0.999188 1 241 0.9994041 248 0.999505 1 250 0.999535 2 260 0.999679 2 262 0.999698 1 2970.999865 1 302 0.999895 1 304 0.999895 2 309 0.999912 2

The fraction (probability) duplex at each position was calculated foreach position according to the probability of a base position containing97% dATP , 100% dCTP, 97% dGTP and 100% dTTP. FIG. 17 (“duplex” curve,left axis) shows the fraction duplex and the frequency of mutation as afunction of sequence position. It is clear that no correlation existsbetween mutational frequency and degree of single stranded overhang. Asshown in the Table 4, there were no mutations found in regions ofhighest single stranded probability (i.e. duplex probabilities <50%) andthe preponderance of mutations occurred at sites that have virtually noprobability of being single stranded (i.e. duplex probability isapproximately 1).

From this data, it is clear that cloning DNA containing significantlengths of single stranded overhang introduces no increased opportunityfor sequence mutation.

Example 8 Generation of an Expression Library

The fact that exposure of amplicon cohesive bases is amplicon sequenceindependent lends this method to generation of expression libraries. 96gene targets from E. coli were cloned using the methods describedherein. 89 successful PCR reactions generated 70 clones displaying 95%or greater sequence homology with the target gene sequence. Thiscorresponds to an approximately 80% success rate neglecting PCR failure,i.e., PCR failure can be due to many factors (primer design, cyclingconditions, solution formulation and etc.) outside of the scope of thecurrent invention. Such a success rate suggests that reliably generatingexpression libraries using the current processes disclosed herein isfeasible.

Example 9 Self Ligation

As demonstrated in FIG. 18, the exonuclease digested amplicons areself-ligatable.

A 500 base pair fragment was amplified from lambda 500 mer DNA using adNTP mixture containing s dATP and s dGTP, and primers specific forBamHI (lane B), EcoRI (lane E), HindIII (lane H), SalI (lane S), XbaI(lane X). The amplicon was digested with ExoIII and followed by ligationat 16° C. for 1 hour. Lane U contains an amplified fragment which wasnot cut with exonuclease III. As shown, all digested amplicons producedligation products demonstrating the premise of self-ligation.

Self ligation would be helpful if one were to attempt to use thismethodology in a gene shuffling or similar combinatorial/molecularevolution experiment. That is, one could take a variety of sequences, donick translation or random priming using the dNTP mixture containingmodified dNTPs, digest with exonucleaseIII then ligate to an adaptor. Itis anticipated that some of the sequences express an enzyme/protein withan altered (improved) property.

Example 10 Ligation of Amplicons to Specifically Designed AdaptorSequences

FIG. 19 outlines the design of adapters designed to add attributes forin vitro translation experiment. Specifically, a 5′ adapter nucleic acidwas designed to contain the T7 promoter and a FLAG® octapeptide codingsequence. The stop codon was engineered into the 3′ adapter. Since thereis a sense strandedness to the design, i.e., ligation of an amplicon tothe adapters orient with the 5′ adapter upstream of a genes sense strandand the stop codon 3′ of the gene's sense strand, ligation to a genefollowed by amplification of the ligation product using adapter basedamplification primers would be most efficient if the ligation proceededdirectionally. Cohesive ends one and two were engineered into the 5′ and3′ adapters respectively for this purpose. Six clones of theseconstructs were sequenced to corroborate that the constructs wereassembled as expected. In each case the ligation orientation was shownto be as expected and without error.

FIG. 20 shows amplification products before (lanes 1, 2) and after(lanes 3, 4) ligation of the above adapter sequence to the digestedamplicons (prepared by s-dATP/s-dGTP containing amplification mixturefollowed by ExoIII digestion) of genes p53 and IkB, respectively.Amplification of the ligation mixture using adapter specific primersclearly yielded amplicons that are longer than those obtained byamplifying with gene specific primers.

It is to be understood that the present invention has been described indetail by way of illustration and example in order to acquaint othersskilled in the art with the invention, its principles, and its practicalapplication. Further, the specific embodiments of the present inventionas set forth are not intended as being exhaustive or limiting of theinvention, and that many alternatives, modifications, and variationswill be apparent to those skilled in the art in light of the foregoingexamples and detailed description. Accordingly, this invention isintended to embrace all such alternatives, modifications, and variationsthat fall within the spirit and scope of the following claims. Whilesome of the examples and descriptions above include some conclusionsabout the way the invention may function, the inventors do not intend tobe bound by those conclusions and functions, but puts them forth only aspossible explanations.

1. An amplicon comprising a double-stranded, amplified nucleic acidfragment, the nucleic acid fragment comprising at least two modifieddNTPs incorporated into the amplicon, wherein the nucleic acid fragmentis resistant to enzymatic degradation by an exonuclease at the site ofincorporation of the at least two modified dNTPs, a first terminuscomplimentary to a first ligation site sequence, and a second terminuscomplimentary to a second ligation site sequence.
 2. The amplicon ofclaim 1 wherein the first and second ligation site sequences are notidentical to each other.
 3. A vector comprising the amplicon of claim 1.4. The amplicon of claim 2 further comprising a first adaptor sequencecomprising a nucleotide sequence encoding for at least one epitope tag.5. The amplicon of claim 4 wherein the at least one epitope tagcomprises c-myc, polyhistidine, polyarginine, glutathione-S-transferase(GST) tag, HA epitope, V5, Xpress™, and FLAG® epitope.
 6. The process ofclaim 5 wherein the at least one epitope tag comprises the FLAG®epitope.
 7. A vector comprising the amplicon of claim
 6. 8. The ampliconof claim 1 wherein the first and second termini of the amplicon are 3′termini.
 9. The amplicon of claim 1 wherein the amplicon has blunt-endedtermini.
 10. The amplicon of claim 1 wherein the amplicon furthercomprises a first single stranded overhang sequence at one 5′ terminusend which is complimentary to the first ligation site sequence, a secondsingle stranded overhang sequence at a second 5′ terminus end which iscomplimentary to the second ligation site sequence.
 11. The amplicon ofclaim 10 wherein the first and second ligation site sequences are notidentical to each other.
 12. The amplicon of claim 10, the ampliconfurther comprising 3′ termini which terminate at sites of incorporationof the modified dNTPs.
 13. The amplicon of claim 12 wherein the firstligation site sequence is an Acc65I, AflII, AgeI, AcaI, ApoI, AvrII,BamHI, BglII, BsiWI, EagI, EcoRI, HindIII, NcoI, NgoMIV, NheI, NotI,SalI, XbaI, XhoI or XmaI recognition sequence, and the second ligationsite sequence is an Acc65I, AflII, AgeI, AcaI, ApoI, AvrII, BamHI,BglII, BsiWI, EagI, EcoRI, HindIII, NcoI, NgoMIV, NheI, NotI, SalI,XbaI, XhoI or XmaI recognition sequence.
 14. The amplicon of claim 12wherein the first ligation site sequence is an EcoRI recognitionsequence.
 15. The amplicon of claim 12 wherein the first ligation sitesequence is an XbaI recognition sequence, and the second ligation sitesequence is a BamHI recognition sequence.